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

Lamichhane, Rajan; Daubner, Gerrit M.; Thomas‐Crusells, Judith; Auweter, Sigrid; Manatschal, Cristina; Austin, Keyunna S.; Valniuk, Oksana; Allain, Frédéric H.‐T.; Rueda, David

doi:10.1073/pnas.0907072107

Cited by 100 publications

(137 citation statements)

References 35 publications

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“…Taken together, these results implicate the KH34 interdomain linker in stabilizing the ZBP1 RNA-binding platform without making direct interactions with the RNA. In contrast to ZBP1 KH34, the third and fourth RRMs of the polypyrimidine tract-binding protein are connected by a long, flexible, polar amino acid-containing linker that has been shown by NMR spectroscopy to directly contact looped segments of exon RNA flanked by RRM-bound pyrimidine tracts (Lamichhane et al 2010). The absence of interactions between the KH34 linker and looped nucleotides suggests that ZBP1 may recognize cellular mRNAs containing both linear arrangements of the REs along a transcript (Fig.…”

Section: Zbp1 Recognizes Its Consensus Rna Elements In Both Linear Armentioning

confidence: 96%

Spatial arrangement of an RNA zipcode identifies mRNAs under post-transcriptional control

Patel¹,

Mitra²,

Harris³

et al. 2012

Genes Dev.

146

196

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How RNA-binding proteins recognize specific sets of target mRNAs remains poorly understood because current approaches depend primarily on sequence information. In this study, we demonstrate that specific recognition of messenger RNAs (mRNAs) by RNA-binding proteins requires the correct spatial positioning of these sequences. We characterized both the cis-acting sequence elements and the spatial restraints that define the mode of RNA binding of the zipcode-binding protein 1 (ZBP1/IMP1/IGF2BP1) to the b-actin zipcode. The third and fourth KH (hnRNP K homology) domains of ZBP1 specifically recognize a bipartite RNA element comprised of a 59 element (CGGAC) followed by a variable 39 element (C/A-CA-C/U) that must be appropriately spaced. Remarkably, the orientation of these elements is interchangeable within target transcripts bound by ZBP1. The spatial relationship of this consensus binding site identified conserved transcripts that were verified to associate with ZBP1 in vivo. The dendritic localization of one of these transcripts, spinophilin, was found to be dependent on both ZBP1 and the RNA elements recognized by ZBP1 KH34.

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Section: Zbp1 Recognizes Its Consensus Rna Elements In Both Linear Armentioning

confidence: 96%

Spatial arrangement of an RNA zipcode identifies mRNAs under post-transcriptional control

Patel¹,

Mitra²,

Harris³

et al. 2012

Genes Dev.

146

196

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show abstract

“…For example, the PTB RRM3 and PTB RRM4 interact via the a helices, which results in the positioning of b-sheet surfaces on opposite sides and complete accessibility for RNA binding (Oberstrass et al 2005). This has functional implications in the mechanism of action of PTB via RNA looping (Lamichhane et al 2010).…”

Section: Novel Features In Cpeb Rrms: Interdomain Interactions and Exmentioning

confidence: 99%

A fly trap mechanism provides sequence-specific RNA recognition by CPEB proteins

et al. 2014

Self Cite

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Cytoplasmic changes in polyA tail length is a key mechanism of translational control and is implicated in germline development, synaptic plasticity, cellular proliferation, senescence, and cancer progression. The presence of a U-rich cytoplasmic polyadenylation element (CPE) in the 39 untranslated regions (UTRs) of the responding mRNAs gives them the selectivity to be regulated by the CPE-binding (CPEB) family of proteins, which recognizes RNA via the tandem RNA recognition motifs (RRMs). Here we report the solution structures of the tandem RRMs of two human paralogs (CPEB1 and CPEB4) in their free and RNA-bound states. The structures reveal an unprecedented arrangement of RRMs in the free state that undergo an original closure motion upon RNA binding that ensures high fidelity. Structural and functional characterization of the ZZ domain (zinc-binding domain) of CPEB1 suggests a role in both protein-protein and protein-RNA interactions. Together with functional studies, the structures reveal how RNA binding by CPEB proteins leads to an optimal positioning of the N-terminal and ZZ domains at the 39 UTR, which favors the nucleation of the functional ribonucleoprotein complexes for translation regulation.

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“…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]. Thus, it appears that the RRMs of PTB may serve not just to recognise specific sequence motifs, but also to bring about structural rearrangements of the RNA.…”

mentioning

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

Cited by 100 publications

References 35 publications

Spatial arrangement of an RNA zipcode identifies mRNAs under post-transcriptional control

Spatial arrangement of an RNA zipcode identifies mRNAs under post-transcriptional control

A fly trap mechanism provides sequence-specific RNA recognition by CPEB proteins

Defining the roles and interactions of PTB

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