Multiple RRMs Contribute to RNA Binding Specificity and Affinity for Polypyrimidine Tract Binding Protein

Abstract

Polypyrimidine tract binding protein (PTB) is a 57 kD hnRNP protein (hnRNP I) that binds to the pyrimidine tract typically found near the 3' end of introns. Primary sequence analysis suggests that PTB contains four RNA recognition motifs (RRMs). Data from comparative structural and deletional analysis of PTB are consistent with the presence of a four reiterated domain structure. Since PTB exists in solution as a homodimer, it contains an oligomeric array of eight RRMs. Though the function of RRMs in a monomeric context has been addressed, the significance of their presence in an oligomeric context has not been investigated. To correlate structural motifs with function, we have analyzed the RNA binding properties of wild-type and deletion constructs of PTB that contain RRMs in both an oligomeric and monomeric context. These studies indicate that there is not a strong correlation between the RNA binding affinity and specificity upon oligomerization. However, the mode of RNA interaction and dimerization is linked. We have also found that the primary contributor to the free energy of PTB binding and the primary determinant for RNA binding specificity resides in RRM 3, while the primary contributor to dimer stabilization coincides with residues in RRM 2.

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

“…PTB is thought to be a nuclear protein in the static state. It can shuttle between the nucleus and cytoplasm, because the RRM 2 domain of PTB facilitates the nuclear export of PTB, whereas RRM 1, 3, and 4 may contribute to the nuclear localization of PTB [46][47][48]. PTB is also an RNA-binding protein; indeed, PTB has been shown to bind to both 5¢UTR [21] …”

A Complex Containing Polypyrimidine Tract-Binding Protein Is Involved in Regulating the Stability of CD40 Ligand (CD154) mRNA

“…49). PTB is known to be a homodimer that belongs to a family of RNA-binding proteins characterized by possession of at least one RNA recognition motif (54) and was originally identified as a nuclear-localized splicing factor that binds to pyrimidine-rich tracts within pre-mRNAs of a large number of genes (Refs. 46, 47, and 55; reviewed in Ref.…”

Polypyrimidine tract binding proteins (PTB) regulate the expression of apoptotic genes and susceptibility to caspase-dependent apoptosis in differentiating cardiomyocytes

“…Similar results were obtained in three independent experiments prepared and transduced a short form of both PTB1 and PTB4 starting at Met175. Short PTB lacked the N-terminal nuclear localization signal (NLS) and the first RRM, which has been shown to influence nuclear shuttling 21,22 and to be dispensable for RNA binding. 21 Unexpectedly, although PTB short forms localized to the cytosol, an important pool was still directed to the nucleus (Figure 5a).…”

“…Short PTB lacked the N-terminal nuclear localization signal (NLS) and the first RRM, which has been shown to influence nuclear shuttling 21,22 and to be dispensable for RNA binding. 21 Unexpectedly, although PTB short forms localized to the cytosol, an important pool was still directed to the nucleus (Figure 5a). PTB short forms retained the capacity to induce nPTB exon 10 skipping ( Figure 5b) and to enhance expression of Apaf-1 and Caspase-3 but were not efficient to sustain Caspase-9, Bax, and Bid expression ( Figure 5c).…”

“…We transduced postnatal cardiomyocytes, which express low levels of endogenous PTB, with several PTB constructs harboring mutations or small deletions of amino acids of the N-terminus, reported to be relevant for nuclear targeting in cell lines. 20,21 However, neither single mutation nor mutation of all key residues altered significantly PTB targeting to the nucleus. This suggested that other regions in PTB are important for nuclear expression in primary cardiomyocytes.…”