Exon repression by polypyrimidine tract binding protein

Amir-Ahmady, Batoul; Boutz, Paul L.; Markovtsov, Vadim; Phillips, Martin; Black, Douglas L.

doi:10.1261/rna.2250405

Cited by 107 publications

(192 citation statements)

References 53 publications

(96 reference statements)

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“…In agreement with previous work, PTB-repressed exons were associated with PTB binding upstream of or within the regulated exon. However, in contrast to some well-studied PTB-repressed exons, including -tropomyosin (Tpm1) exon 3 [14,15] and SRC N1 exon [16], we found no general tendency for PTB binding downstream of repressed exons. In contrast, the newly characterized PTB-activated exons were clearly associated with PTB binding on the downstream side of the exon [12].…”

Section: Ptb Splicing Mapscontrasting

confidence: 99%

“…To fully understand how PTB functions, it is important to know how many PTB molecules bind to target RNAs. Conventional biochemical assays, such as electrophoretic mobility shift or filter-binding, have shown that splicing substrates typically bind more than one PTB molecule [14][15][16]32]. However, these analyses have used pure recombinant PTB, which could easily lead to overestimates of the true number of molecules bound in vivo, where non-specific binding will be suppressed by the numerous other proteins that coat the RNA.…”

Section: Counting Proteins In Complexesmentioning

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: Ptb Splicing Mapscontrasting

confidence: 99%

Section: Counting Proteins In Complexesmentioning

confidence: 99%

Defining the roles and interactions of PTB

Kafasla

Mickleburgh

Llorian

et al. 2012

Biochemical Society Transactions

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“…DNA Constructs. PTB1, nPTB, and reporter plasmid DS9-175 were prepared as described (26). The three mutations in PTB1 (PTB 3Mut) were made with two consecutive PCR reactions using the primers 2Mut-F, 2Mut-R, 1Mut-F, and 1Mut-R (Table S1).…”

Section: Methodsmentioning

confidence: 99%

“…To test how functionally important is the interface between RRM3 and RRM4, we used a splicing reporter assay (25) in HeLa cells with a reporter gene containing two separate PPTs separated by a long linker (DS9-175) (26). In a control experiment without protein over expression, the alternative exon is 80% included, whereas only 34% is included when PTB is transfected and overexpressed.…”

Section: Rna Looping By Ptb34 Is Essential For Efficient Splicing Regmentioning

confidence: 99%

RNA looping by PTB: Evidence using FRET and NMR spectroscopy for a role in splicing repression

Lamichhane

Daubner²,

Thomas‐Crusells³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

100

123

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Alternative splicing plays an important role in generating proteome diversity. The polypyrimidine tract-binding protein (PTB) is a key alternative splicing factor involved in exon repression. It has been proposed that PTB acts by looping out exons flanked by pyrimidine tracts. We present fluorescence, NMR, and in vivo splicing data in support of a role of PTB in inducing RNA loops. We show that the RNA recognition motifs (RRMs) 3 and 4 of PTB can bind two distant pyrimidine tracts and bring their 5′ and 3′ ends in close proximity, thus looping the RNA. Efficient looping requires an intervening sequence of 15 nucleotides or longer between the pyrimidine tracts. RRM3 and RRM4 bind the 5′ and the 3′ pyrimidine tracts, respectively, in a specific directionality and work synergistically for efficient splicing repression in vivo.alternative splicing | polypyrimidine tract-binding protein | protein-RNA interactions A lternative splicing is a highly regulated biological process that plays a crucial role in generating high proteomic diversity. It has been estimated that >90% of human genes are alternatively spliced (1). Alternative splicing occurs frequently in cells, and most RNA-binding proteins that influence alternative splicing were found to be nonspliceosomal (2). The polypyrimidine tract (PPT)-binding protein (PTB) is one of the major trans-acting factors involved in splicing regulation. PTB is most often associated with its role as a splicing repressor (3-5), but it is also involved in other aspects of mRNA processing including 3′ end processing (6, 7), mRNA localization and stability (8), and internal ribosome entry site (IRES)-mediated translation (9).PTB is a 58-kDa member of the hnRNP family consisting of four RNA recognition motifs (RRMs) joined by three linkers (10, 11). PTB recognizes PPTs in the RNA target containing CU-rich elements (12, 13). The mechanism by which PTB promotes exon exclusion is poorly understood. Our NMR structure of RNAbound PTB has suggested a potential mechanism of PTB action in splicing whereby RRM3 and RRM4 bind the PPTs flanking an alternative exon and loop out the intervening RNA, thus repressing the exon (Fig. 1A) (14). The two RRM-bound PPTs appear in opposite direction as if forming a loop to exclude the intervening exon or the branched adenosine from the spliceosomal machinery. Other mechanistic models for PTB repression have proposed a direct (5) and an indirect (5, 15) competition between PTB and other splicing factors like U2AF65, corepression with Raver-1 (16) and PTB preventing exon (15) or intron definition (17). However, all proposed mechanisms are consistent with RNA looping between RRM3 and RRM4.Here, we have sought to test and characterize this suggested looping mechanism using FRET, NMR spectroscopy, and in vivo splicing assays. Results PTB34 Binds PPTs and Brings Their 5′ and 3′ Ends into Close Proximity.First, we tested the binding of RRM3 and RRM4 of PTB (PTB34, Fig. 1A) to several model RNAs using a FRET-based gel shift assay (18). We prepared a series of singl...

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“…PTB is one of the most frequently encountered ITAFs and was found to be implicated in IRES-mediated translation of several Figure 1. Polypyrimidine tract binding protein (PTB) is a ubiquitous regulator of alternative splicing that influences different types of alternative-splicing events (a) PTB represses the N1 "cassette-exon" in the c-src pre-mRNA [7,9,10,103]. (b) PTB represses the "mutually exclusive" SM exon of the a-actinin mRNA [12,13].…”

Section: The Many Functions Of Polypyrimidine Tract Binding Proteinmentioning

confidence: 99%

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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Exon repression by polypyrimidine tract binding protein

Cited by 107 publications

References 53 publications

Defining the roles and interactions of PTB

Defining the roles and interactions of PTB

RNA looping by PTB: Evidence using FRET and NMR spectroscopy for a role in splicing repression

Structure-function relationships of the polypyrimidine tract binding protein

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