Abstract
The polypyrimidine tract binding protein (PTB) is a multi-domain protein involved in alternative splicing, mRNA localization, stabilization, polyadenylation and translation initiation from internal ribosome entry sites (IRES). In this latter process, PTB promotes viral translation by interacting extensively with complex structured regions in the 5′-untranslated regions of viral RNAs at pyrimidine-rich targets located in single strand and hairpin regions. To better understand how PTB recognizes structured elements in RNA targets, we solved the solution structure of the N-terminal RNA recognition motif (RRM) in complex with an RNA hairpin embedding the loop sequence UCUUU, which is frequently found in IRESs of the picornovirus family. Surprisingly, a new three-turn α3 helix C-terminal to the RRM, folds upon binding the RNA hairpin. Although α3 does not mediate any contacts to the RNA, it acts as a sensor of RNA secondary structure, suggesting a role for RRM1 in detecting pyrimidine tracts in the context of structured RNA. Moreover, the degree of helix formation depends on the RNA loop sequence. Finally, we show that the α3 helix region, which is highly conserved in vertebrates, is crucial for PTB function in enhancing Encephalomyocarditis virus IRES activity.
Highlights
RNA binding proteins (RBPs) are essential in the regulation of diverse processes in RNA biology, such as mRNA splicing, RNA transport, storage, degradation, post-transcriptional modification and translation
We investigated by NMR titration the binding of PTB RRM1 to a 23 nucleotide RNA (Figure 2A) that embeds the UCUUU apical loop sequence found in SL F and SL H of encephalomyocarditis virus (EMCV) internal ribosome entry sites (IRES), and in stem–loops of other picornavirus IRES (Figure 1B)
SL UCUUU formed a single hairpin conformation in solution under our NMR conditions with standard Watson-Crick base-pairing indicated for the stem by an imino walk in 2D NOESY spectra and an imino–imino NOE detected between the uridines at the 5 and 3 end of the loop
Summary
RNA binding proteins (RBPs) are essential in the regulation of diverse processes in RNA biology, such as mRNA splicing, RNA transport, storage, degradation, post-transcriptional modification and translation. PTB acts primarily as a repressive splicing regulator It can enhance exon inclusion and the role it plays depends on the relative position of its binding site, exons and the polyadenylation signal [5,6,7,8]. PTB interacts in particular with a number of viral IRES RNAs from the picornoviridae family, which comprises poliovirus (PV), human rhinovirus (HRV), hepatitis A virus (HAV), foot and mouth disease virus (FMDV), Theiler’s murine encephalomyelitis virus (TMEV) and encephalomyocarditis virus (EMCV). These IRES RNAs adopt highly complex structures, which contain short and long pyrimidine stretches identified as PTB binding sites. It has been proposed that PTB plays the role of an RNA chaperone and that it may stabilize or rearrange IRES RNA structure in or-
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