Structure of PTB Bound to RNA: Specific Binding and Implications for Splicing Regulation

Oberstrass, Florian C.; Auweter, Sigrid; Erat, Michèle C.; Hargous, Yann; Henning, A; Wenter, Philipp; Reymond, Luc; Amir-Ahmady, Batoul; Pitsch, Stefan; Black, Douglas L.; Allain, Frédéric H.‐T.

doi:10.1126/science.1114066

Cited by 424 publications

(701 citation statements)

References 30 publications

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“…5a). The unusual orientation of the C-terminal loop and α-helix of Snu17p within the RNA-binding site suggests that the RES-RNA interaction is potentially another example of a noncanonical RRM-RNA recognition mode [38][39][40][49][50][51][52][53] . The increase in RNA affinity when c Pml1p is bound to c Snu17p and the C-terminal α-helix of Snu17p is formed (Supplementary Fig.…”

Section: Discussionmentioning

confidence: 99%

Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex

Wysoczanski

Schneider

Munari

et al. 2014

Nat Struct Mol Biol

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1 1 a r t i c l e sSplicing entails the removal of introns from pre-mRNAs to generate exon-only mRNA, which is exported out of the nucleus for translation 1 . The splicing process is driven and controlled by a large and dynamic RNA-protein complex, the spliceosome 1,2 . The composition and structure of the spliceosome undergoes multiple rearrangements during a splicing reaction, in which a set of distinct structural and compositional states, designated as E, A, B act , B* and C complexes, can be defined 1 . As part of this process, small nuclear ribonucleoprotein (snRNP) particles and non-snRNP splice factors are recruited and released 1,2 . Although the organization of snRNP components of the spliceosome has received considerable attention in recent years, very little is known about the assembly, structure and dynamics of non-snRNP multimeric complexes.The non-snRNP RES complex is present in humans and yeast 3,4 . Deletion of RES genes slows splicing and leads to pre-mRNA leakage into the cytoplasm 3-5 . Distinct introns exhibit RES-dependent splicing 5-9 , as do pre-mRNAs encoding proteins functioning in RNA-nucleotide metabolism 8,10 . Components of the RES complex are found in B and C complexes of the spliceosome, in which RES can interact with U2 snRNP 4,11,12 . In yeast, the RES complex is composed of three proteins, snRNP-associated protein 17 (Snu17p, also known as Ist3p), pre-mRNA-leakage protein 1 (Pml1p) and bud siteselection protein 13 (Bud13p) 3,4 . Snu17p and Bud13p have been implicated directly in splicing 3,5,13 , whereas Pml1p has been linked to the retention of unspliced pre-mRNA in the nucleus 3,5 . Caenorhabditis elegans Bud13p is involved in embryogenesis 14 .Sequence analysis has indicated that Snu17p is a 148-residue (17.1-kDa) noncanonical member of the RRM family of proteins with a long C-terminal part, which exhibits low sequence similarity to published RRM structures 4 . Snu17p binds with nanomolar affinity to the 266-residue (30.5-kDa), natively disordered protein Bud13p 15 . The interaction has been postulated to involve a C-terminal UHM-ligand motif (ULM) in Bud13p that interacts with a U2AF-homology motif (UHM) in the RRM domain of Snu17p 13,15,16 . The only other identified domain encompasses a stretch of lysine residues at the N terminus of Bud13p. Binding of the third component, the 204-residue (23.4-kDa) Pml1p, occurs through its 50 N-terminal disordered residues. The remainder of Pml1p folds as a forkhead-associated domain 13,15,17 , which could potentially bind phosphopeptides 17 . Biochemical evidence has suggested that Snu17p acts as the central binding platform, which interacts with disordered parts of Bud13p and Pml1p 13,15,16 . The precise molecular architecture of the RES complex, however, has been elusive, and its RNA binding capabilities have remained unexplored.Here we solved the three-dimensional structure of the core of the RES complex and demonstrated that its assembly is driven by cooperativity that increases the binding affinity of the components of the complex ...

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Section: Discussionmentioning

confidence: 99%

Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex

Wysoczanski

Schneider

Munari

et al. 2014

Nat Struct Mol Biol

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“…PTBP1 serves as a repressor of alternative splicing in mammalian cells (24)(25)(26)(27)(28)(29) and contains RNA-binding domains, each of which binds to CU-rich elements (30). It is involved in polyadenylation of the pre-mRNA 3 0 end (31-33) and also plays an important role in translational regulation of a subset of RNA transcription through internal ribosome entry sites (21,(34)(35)(36)(37).…”

Section: Discussionmentioning

confidence: 99%

Significance of Polypyrimidine Tract–Binding Protein 1 Expression in Colorectal Cancer

Takahashi

Nishimura

Kagawa

et al. 2015

Molecular Cancer Therapeutics

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Polypyrimidine tract-binding protein (PTBP1) is an RNAbinding protein with various molecular functions related to RNA metabolism and a major repressive regulator of alternative splicing, causing exon skipping in numerous alternatively spliced pre-mRNAs. Here, we have investigated the role of PTBP1 in colorectal cancer. PTBP1 expression levels were significantly overexpressed in cancerous tissues compared with corresponding normal mucosal tissues. We also observed that PTBP1 expression levels, c-MYC expression levels, and PKM2: PKM1 ratio were positively correlated in colorectal cancer specimens. Moreover, PTBP1 expression levels were positively correlated to poor prognosis and lymph node metastasis. In analyses of colorectal cancer cells using siRNA for PTBP1, we observed that PTBP1 affects cell invasion, which was partially correlated to CD44 splicing, and this correlation was also confirmed in clinical samples. PTBP1 expression also affected anchorage-independent growth in colorectal cancer cell lines. PTBP1 expression also affected cell proliferation. Using timelapse imaging analysis, PTBP1 was implicated in prolonged G 2 -M phase in HCT116 cells. As for the mechanism of prolonged G 2 -M phase in HCT116 siPTBP1 cells, Western blotting revealed that PTBP1 expression level was correlated to CDK11 p58 expression level, which was reported to play an important role on progression to complete mitosis. These findings indicated that PTBP1 is a potential therapeutic target for colorectal cancer.

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“…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].…”

Section: Functional Domains Of Ptbmentioning

confidence: 99%

“…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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Structure of PTB Bound to RNA: Specific Binding and Implications for Splicing Regulation

Cited by 424 publications

References 30 publications

Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex

Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex

Significance of Polypyrimidine Tract–Binding Protein 1 Expression in Colorectal Cancer

Defining the roles and interactions of PTB

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