Defining the roles and interactions of PTB

Abstract

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...

The Stress Granule Component TIA-1 Binds Tick-Borne Encephalitis Virus RNA and Is Recruited to Perinuclear Sites of Viral Replication To Inhibit Viral Translation

“…To address whether this relocalization was a specific feature of TIA-1, we choose the PTB protein, which has been implicated in the life cycles of DENV and JEV (28)(29)(30). PTB shares several features with TIA-1, since both proteins (i) are RNA-binding proteins with several RNA recognition motifs; (ii) are shuttling proteins with a predominant nuclear localization; (iii) are involved in nuclear processes, such as alternative splicing; and (iv) are involved in cytoplasmic processes, such as translation (31). As shown in Fig.…”

Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1

“…The polypyrimidine tract (PT) is another important cis-acting element that impacts splicing [23]. The PT consensus sequence (Fig.…”

Regulation of Alternative Splicing Through Coupling with Transcription and Chromatin Structure

“…The former include exonic splicing enhancers, exonic splicing silencers, intronic splicing enhancers, and intronic splicing silencers, depending on their locations and on how they affect the usage of a splice site. Trans-acting factors work through binding to splicing enhancers and silencers and include members of well-characterized serine-arginine (SR)-rich and heterogeneous nuclear ribonucleoprotein (hnRNP) families of proteins, as well as tissue-specific factors, such as polypyrimidine tract-binding protein (PTB) (9), NOVA (10), and FOX (11). Some of these factors activate, whereas others inhibit, the use of splice sites.…”