Polypyrimidine Tract Binding Protein Stabilizes the Encephalomyocarditis Virus IRES Structure via Binding Multiple Sites in a Unique Orientation

Abstract

Polypyrimidine tract binding (PTB) protein is a regulator of alternative pre-mRNA splicing, and also stimulates the initiation of translation dependent on many viral internal ribosome entry segments/sites (IRESs). It has four RNA-binding domains (RBDs), but although the contacts with many IRESs have been mapped, the orientation of binding (i.e., which RBD binds to which site in the IRES) is unknown. To answer this question, 16 derivatives of PTB1, each with a single cysteine flanking the RNA-binding surface in an RBD, were constructed and used in directed hydroxyl radical probing with the encephalomyocarditis virus IRES. The results, together with mass spectrometry data on the stoichiometry of PTB binding to different IRES derivatives, show that the minimal IRES binds a single PTB in a unique orientation, with RBD1 and RBD2 binding near the 3' end, and RBD3 contacting the 5' end, thereby constraining and stabilizing the three-dimensional structural fold of the IRES.

Picornavirus IRES elements: RNA structure and host protein interactions

“…Several picornavirus IRES elements have two polypyrimidine tracts located at each end of the IRES region. In agreement with the functional role of each pyrimidine tract on IRES activity it has been found that PTB constrains the EMCV IRES structure in a unique orientation by binding to the RNA with RRM1-2 contacting the 3 end, and RRM3 contacting the 5 end of the IRES (Kafasla et al, 2009).…”

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

“…This parent DNTD-PTB construct, hereafter designated PTB9, also has an N-terminal His-tag, and when the two remaining cysteine residues (C250 and C251 in RBD2) were replaced by serines (Fig. 1C), it resulted in the Cys-less PTB9 construct which is used as the control in tethered hydroxyl radical probing assays of the interaction of each RBD with the target RNA, as in our previous work (Kafasla et al 2009(Kafasla et al , 2010.…”

“…To this end, the point mutations in the RNA-binding surface of a particular RBD were initially put into the background of a single cysteine derivative where the cysteine is located in the same RBD. From our library of 16 single cysteine PTB9 derivatives which all reacted efficiently with Fe(II)-BABE (Kafasla et al 2009), we selected six for these assays-one in each of RBDs2 and 3 and two in each of RBDs1 and 4 (Fig. 1C).…”

“…However, these approaches provide no information on the orientation of the PTB binding to its target RNAs-the question of which RBD binds to which site on the RNA. We have recently generated a battery of PTB mutants, each with a single cysteine positioned at sites flanking the actual RNA-binding surface of each RBD, and we have used these derivatives in tethered hydroxyl radical probing to map the orientation of PTB binding to the EMCV and poliovirus IRESs (Kafasla et al 2009(Kafasla et al , 2010. The results showed a very different pattern of PTB interaction with these two IRESs.…”

“…The results showed a very different pattern of PTB interaction with these two IRESs. The core EMCV IRES bound a single PTB, with RBDs1 and 2 binding near the 39-end of the IRES and RBDs3 and 4 interacting with the 59-end of the core IRES (Kafasla et al 2009). This suggested that PTB binding would constrain the flexibility of the three-dimensional structure of the IRES and probably help to stabilize the optimum structure for internal initiation.…”