Structure of the two most C-terminal RNA recognition motifs of PTB using segmental isotope labeling

Vitali, Francesca; Henning, A; Oberstrass, Florian C.; Hargous, Yann; Auweter, Sigrid; Erat, Michèle C.; Allain, Frédéric H.‐T.

doi:10.1038/sj.emboj.7600911

Cited by 91 publications

(142 citation statements)

References 56 publications

(87 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%

“…1A), as well as interesting features of RRM organization. Notably, while the linkers between RRMs 1, 2 and 3 are flexible [23], RRMs 3 and 4 form a stable di-domain with back to back packing of the two RRMs involving the short linker [20,22]. This di-domain structure necessitates a loop of at least 15 nt between the two pyrimidine tracts recognized by RRMs 3 and 4; targeted mutations to disrupt the didomain packing impair PTBs regulatory activity on SRC splicing [24].…”

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confidence: 99%

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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%

mentioning

confidence: 99%

Defining the roles and interactions of PTB

Kafasla

Mickleburgh

Llorian

et al. 2012

Biochemical Society Transactions

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“…Measurements of protein dynamics, as well as a mutational analysis, could further confirm the interaction. As many as 20 side chains that lie in both helices of RRM3, in helix 2 of RRM4 and in the interdomain linker form a hydrophobic cluster that glues the two domains together so that their RNA binding interfaces point in opposite directions [80]. This large interaction between two RRMs is very unusual among RRMs in their free state, the only other example being the recent structure of Prp24, a protein containing three RRMs [81].…”

mentioning

confidence: 99%

“…While the hydrogen bond network present in RRM1 predicts that any YCU sequence can be bound, RRM2, RRM3 and RRM4 specifically recognize CU(N)N, YCUNN and YCN, respectively [85] (where Y stands for pyrimidine and N for any nucleotide). Finally, the overall fold of the PTB RRMs is identical in free [79,80,82] and RNA-bound forms [85] (Figs. 3 and 4).…”

mentioning

confidence: 99%

“…Initially, RRM3 and 4 of PTB were suggested to be independent based on the lack of interdomain NOEs between the domains [79]. However, a subsequent structural characterization of the same construct revealed that this was not true [80]. In this second NMR study, segmental isotope labeling of the two domains was employed to obtain a large number of inter-domain distance constraints and demonstrate unambiguously the extensive interdomain interaction [80] (Fig.…”

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confidence: 99%

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Structure-function relationships of the polypyrimidine tract binding protein

Auweter

Allain

2007

Cell. Mol. Life Sci.

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Abstract. The polypyrimidine tract binding protein (PTB) is a 58-kDa RNA binding protein involved in multiple aspects of mRNA metabolism including splicing regulation, polyadenylation, 3'end formation, internal ribosomal entry site-mediated translation, RNA localization and stability. PTB contains four RNA recognition motifs (RRMs) separated by three linkers. In this review we summarize structural information on PTB in solution that has been gathered during the past 7 years using NMR spectroscopy and small-angle X-ray scattering. The structures of all RRMs of PTB in their free state and in complex with short pyrimidine tracts, as well as a structural model of PTB RRM2 in complex with a peptide, revealed unusual structural features that provided new insights into the mechanisms of action of PTB in the different processes of RNA metabolism and in particular splicing regulation.

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