Structure and RNA Interactions of the N-Terminal RRM Domains of PTB

Simpson, Peter; Monie, Tom P.; Szendroi, A.; Davydova, Natalia; Tyzack, Jonathan K.; Conte, Maria R.; Read, Christopher M.; Cary, Peter D.; Svergun, Dmitri I.; Konarev, Petr V.; Curry, Stephen; Matthews, Stephen

doi:10.1016/j.str.2004.07.008

Cited by 92 publications

(161 citation statements)

References 57 publications

(2 reference statements)

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“…Although full-length PTB has eluded high resolution structural determination, the structures of all four PTB RRMs have been determined by NMR, in both free form as well as bound to a hexameric CUCUCU RNA ligand [19][20][21][22]. This has revealed the basis of specificity of RNA recognition by each RRM (Fig.…”

Section: Functional Domains Of Ptbmentioning

confidence: 99%

Defining the roles and interactions of PTB

Kafasla

Mickleburgh

Llorian

et al. 2012

Biochemical Society Transactions

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Polypyrimidine tract binding (PTB) protein is an abundant and widely expressed RNA binding protein with four RNA Recognition Motif (RRM) domains. PTB is involved in numerous post-transcriptional steps in gene expression in both the nucleus and cytoplasm, but has been bestcharacterized as a regulatory repressor of some alternative splicing events (ASEs), and as an activator of translation driven by internal ribosome entry segments (IRESs). We have used a variety of approaches to characterize the activities of PTB and its molecular interactions with RNA substrates and protein partners. Using splice-sensitive microarrays we found that PTB acts not only as a splicing repressor but also as an activator, and that these two activities are determined by the location at which PTB binds relative to target exons. We have identified minimal splicing repressor and activator domains, and have determined high resolution structures of the second RRM domain of PTB binding to peptide motifs from the co-repressor protein Raver1. Using single-molecule techniques we have determined the stoichiometry of PTB binding to a regulated splicing substrate in whole nuclear extracts. Finally, we have used tethered hydroxyl radical probing to determine the locations on viral IRESs at which each of the four RRM domains bind. We are now combining tethered probing with single molecule analyses to gain a detailed understanding of how PTB interacts with pre-mRNA substrates to effect either repression or activation of splicing. 3Polypyrimidine tract binding protein (PTB) is an abundant RNA binding protein of the hnRNP family, originally identified by its binding to the polypyrimidine tract at the 3´ splice site of mammalian introns [1, 2]. Structurally, PTB consists of four RNA Recognition Motif (RRM) domains [3], with three interdomain linkers and an N-terminal leader sequence containing nuclear localization and export signals (Fig. 1A). Early speculation that PTB was an essential pre-mRNA splicing factor was rapidly dispelled, and it was subsequently recognized as a repressive regulator of alternative splicing [4]. In vitro selection experiments showed that optimal binding substrates for PTB consisted of motifs such as UCUUC embedded within more extended pyrimidine-rich contexts [5, 6]. Such motifs were found in splicing silencer elements associated with many PTB repressed exons (Fig. 1B). PTB was also found to be involved in regulating numerous other post-transcriptional processes in both the nucleus and cytoplasm, including 3´-end processing, mRNA stability, internal ribosome entry segment (IRES) driven translation and mRNA localization [7]. Of these processes, most experimental attention has focused on the roles of PTB as a repressor of splicing and activator of IRESmediated translation initiation. Here we discuss a range of approaches that we have deployed recently to analyze the roles of PTB and its interactions with RNA targets and protein partners. PTB splicing mapsPTB has been investigated as a splicing repressor in numerous mod...

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Section: Functional Domains Of Ptbmentioning

confidence: 99%

Defining the roles and interactions of PTB

Kafasla

Mickleburgh

Llorian

et al. 2012

Biochemical Society Transactions

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“…RBDs contribute to the binding of PTB to its target RNAs (Simpson et al 2004;Oberstrass et al 2005), and, in particular, the finding that deletion mutants which retain just two RBDs have significantly lower binding affinity than full-length PTB for the EMCV IRES (and also for a pre-mRNA target of PTB), with the affinity of the RBD3 + 4 combination distinctly lower than that of the RBD1 + 2 pair (Simpson et al 2004).…”

Section: Mechanism Of Picornavirus Iresmentioning

confidence: 99%

“…PTB is a monomer in solution, but it has an extended conformation (Simpson et al 2004;Monie et al 2005), with flexible linkers between RBDs 1 and 2 and between RBDs 2 and 3. In contrast, RBDs 3 and 4 are separated by a short linker and seem to act as a pair or didomain (Oberstrass et al 2005).…”

Section: Introductionmentioning

confidence: 99%

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

Kafasla¹,

Lin²,

Curry³

et al. 2011

RNA

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Polypyrimidine tract binding protein (PTB) is an RNA-binding protein with four RNA-binding domains (RBDs). It is a major regulator of alternative splicing and also stimulates translation initiation at picornavirus IRESs (internal ribosome entry sites). The sites of interaction of each RBD with two picornaviral IRESs have previously been mapped. To establish which RBD-IRES interactions are essential for IRES activation, point mutations were introduced into the RNA-binding surface of each RBD. Three such mutations were sufficient to inactivate RNA-binding by any one RBD, but the sites of the other three RBD-IRES interactions remained unperturbed. Poliovirus IRES activation was abrogated by inactivation of RBD1, 2, or 4, but the RBD3-IRES interaction was superfluous. Stimulation of the encephalomyocarditis virus IRES was reduced by inactivation of RBD1, 3, or 4, and abrogated by mutation of RBD2, or both RBDs 3 and 4. Surprisingly, therefore, the binding of PTB in its normal orientation does not guarantee IRES activation; three native RBDs are sufficient for correct binding but not for activation if the missing RBD-IRES interaction is critical.

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“…42 However, recent data suggest that all four RRMs contribute to the RNA binding of PTB, and that PTB binds to its target RNA as an 'extended monomer'. 43 There are two well-characterised examples of PTB affecting IRES-mediated initiation of proteins that have roles in the apoptotic process; both the Apaf-1 and BAG-1 IRESs require PTB for function. [44][45][46][47] In fact the Apaf-1 IRES requires two trans-acting factors for function.…”

Section: Analysis Of Mrnas That Remain Polysomally Associated During mentioning

confidence: 99%

Internal ribosome entry segment-mediated translation during apoptosis: the role of IRES-trans-acting factors

et al. 2005

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During apoptosis, there is a reduction in translation initiation caused by caspase cleavage of several of the factors required for the cap-dependent scanning mechanism. Under these circumstances, many proteins that are required for apoptosis are instead translated by the alternative method of internal ribosome entry. This mechanism requires the formation of a complex RNA structural element and in the presence of internal ribosome entry segment (IRES)-trans-acting factors (ITAFs), the ribosome is recruited to the RNA. The interactions of several ITAFs with IRESs have been investigated in detail, and several mechanisms of action have been noted, including acting as chaperones, stabilising and remodelling the RNA structure. Structural remodelling by PTB in particular will be discussed, and how this protein is able to facilitate recruitment of the ribosome to several IRESs by causing previously occluded sites to become more accessible.

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Structure and RNA Interactions of the N-Terminal RRM Domains of PTB

Cited by 92 publications

References 57 publications

Defining the roles and interactions of PTB

Defining the roles and interactions of PTB

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

Internal ribosome entry segment-mediated translation during apoptosis: the role of IRES-trans-acting factors

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