A transient α-helix in the N-terminal RNA recognition motif of polypyrimidine tract binding protein senses RNA secondary structure

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.

Sequence-specific RNA recognition by an RGG motif connects U1 and U2 snRNP for spliceosome assembly

“…In addition, based on our recent structural investigation of the RNA binding properties of the N-terminal RRM (RNA recognition motif) of PTBP1 bound to a pyrimidine-rich RNA pentaloop, we proposed that PTBP1-RRM1 would bind the UUCG tetraloop of U1-SL4 from the major-groove side (Fig. 6C) (26), similar to what we observed here for SF3A1-UBL. Since PTBP1 was previously shown to bind U1-SL4 to inhibit splicing (13), our structure reveals that the binding of PTBP1 and SF3A1 could be mutually exclusive, and therefore, competition between the two proteins may regulate splicing (see Discussion).…”

Integrative solution structure of a PTBP1-viral IRES complex reveals strong compaction and ordering with residual conformational flexibility

“…2D). This proximity is facilitated by the 10 residues C-terminal to RRM1 that fold into an α3-helix upon binding to SLE, reducing the overall length and dynamics of the linker (38). Close examination of the 25 conformers revealed that RRM1 may favor conformations placing it close to RRM2 because the inter-domain linker is very hydrophobic and folds back as a hairpin, resulting in RRM1 facing the β-sheet of RRM2 (seen in models 1-7) (Fig.…”

“…An increase in flexibility of the complex upon mutation of the RRM4 binding site in LinkEF coincides with a dramatic reduction in translation efficiency. Third, binding of RRM1 to SLE results in a shortening of the linker between RRM1 and RRM2 (38). Fourth, there is a close interaction between RRM1 and RRM2 that is mediated by several sets of interactions, and not a unique one.…”

PTB: Not just a polypyrimidine tract‐binding protein

“…However, recent studies have shown that the tandem RRM34 prefers single stranded RNA sites, RRM12 can also bind to RNA, and full‐length PTB is a monomer (Monie et al, 2005; Robinson & Smith, 2006). Maris et al (2020) determined the structure of the RRM1 in complex with an RNA hairpin containing a UCUUU apical loop which folds upon binding the RNA hairpin acts as a sensor of the RNA secondary structure. Marnef et al (2016) demonstrated PTB forms a direct interaction with the T‐loop and the d ‐stem‐loop of mitochondrial (mt) tRNA (Thr) using its N‐terminal RRM1 and RRM2 motifs.…”