Structure-function relationships of the polypyrimidine tract binding protein

Auweter, Sigrid; Allain, Frédéric H.‐T.

doi:10.1007/s00018-007-7378-2

Cited by 80 publications

(89 citation statements)

References 97 publications

(141 reference statements)

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“…3A). This finding is consistent with earlier studies showing that RNA binding-defective PTB proteins generally result from mutations located in the conserved RNA recognition motifs (45). We next tested whether any of the CmRBP50-interacting proteins had RNA-binding activity.…”

Section: Resultssupporting

confidence: 90%

CmRBP50 Protein Phosphorylation Is Essential for Assembly of a Stable Phloem-mobile High-affinity Ribonucleoprotein Complex

Ham

Lucas

2011

Journal of Biological Chemistry

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RNA-binding proteins (RBPs) form ribonucleoprotein (RNP)complexes that play crucial roles in RNA processing for gene regulation. The angiosperm sieve tube system contains a unique population of transcripts, some of which function as long-distance signaling agents involved in regulating organ development. These phloem-mobile mRNAs are translocated as RNP complexes. One such complex is based on a phloem RBP named Cucurbita maxima RNA-binding protein 50 (CmRBP50), a member of the polypyrimidine track binding protein family. The core of this RNP complex contains six additional phloem proteins. Here, requirements for assembly of this CmRBP50 RNP complex are reported. Phosphorylation sites on CmRBP50 were mapped, and then coimmunoprecipitation and protein overlay studies established that the phosphoserine residues, located at the C terminus of CmRBP50, are critical for RNP complex assembly. In vitro pull-down experiments revealed that three phloem proteins, C. maxima phloem protein 16, C. maxima GTP-binding protein, and C. maxima phosphoinositide-specific phospholipase-like protein, bind directly with CmRBP50. This interaction required CmRBP50 phosphorylation. Gel mobility-shift assays demonstrated that assembly of the CmRBP50-based protein complex results in a system having enhanced binding affinity for phloem-mobile mRNAs carrying polypyrimidine track binding motifs. This property would be essential for effective long-distance translocation of bound mRNA to the target tissues.

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Section: Resultssupporting

confidence: 90%

CmRBP50 Protein Phosphorylation Is Essential for Assembly of a Stable Phloem-mobile High-affinity Ribonucleoprotein Complex

Ham

Lucas

2011

Journal of Biological Chemistry

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“…All the mutants were made in the background of a PTB-1 construct with a deletion of the N-terminal domain (NTD) (amino acids 1-54), which has nuclear import and export signals but is not involved in the interaction of PTB with RNA (Auweter and Allain 2008). This parent DNTD-PTB construct, hereafter designated PTB9, also has an N-terminal His-tag, and when the two remaining cysteine residues (C250 and C251 in RBD2) were replaced by serines (Fig.…”

Section: Mutants Generatedmentioning

confidence: 99%

“…Polypyrimidine tract binding protein (PTB) has four RNAbinding domains (RBDs), also known as RRMs (RNA recognition motif), of the RNP1/RNP2 class (Oberstrass et al 2005;Auweter and Allain 2008), although these elements are rather atypical in their lack of aromatic residues. PTB is a monomer in solution, but it has an extended conformation (Simpson et al 2004;Monie et al 2005), with flexible linkers between RBDs 1 and 2 and between RBDs 2 and 3.…”

Section: Introductionmentioning

confidence: 99%

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Activation of picornaviral IRESs by PTB shows differential dependence on each PTB RNA-binding domain

Kafasla¹,

Lin²,

Curry³

et al. 2011

RNA

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Polypyrimidine tract binding protein (PTB) is an RNA-binding protein with four RNA-binding domains (RBDs). It is a major regulator of alternative splicing and also stimulates translation initiation at picornavirus IRESs (internal ribosome entry sites). The sites of interaction of each RBD with two picornaviral IRESs have previously been mapped. To establish which RBD-IRES interactions are essential for IRES activation, point mutations were introduced into the RNA-binding surface of each RBD. Three such mutations were sufficient to inactivate RNA-binding by any one RBD, but the sites of the other three RBD-IRES interactions remained unperturbed. Poliovirus IRES activation was abrogated by inactivation of RBD1, 2, or 4, but the RBD3-IRES interaction was superfluous. Stimulation of the encephalomyocarditis virus IRES was reduced by inactivation of RBD1, 3, or 4, and abrogated by mutation of RBD2, or both RBDs 3 and 4. Surprisingly, therefore, the binding of PTB in its normal orientation does not guarantee IRES activation; three native RBDs are sufficient for correct binding but not for activation if the missing RBD-IRES interaction is critical.

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“…Solution structures of each PTBP1 RRM bound to RNA identified residues important for RNA recognition and binding (Conte et al 2000;Simpson et al 2004;Oberstrass et al 2005;Petoukhov et al 2006;Auweter and Allain 2008). PTBP2 has the same residues at these positions of RNA contact except for one lysine to arginine and two phenylalanine to tyrosine substitutions (Fig.…”

Section: Introductionmentioning

confidence: 98%

Multiple determinants of splicing repression activity in the polypyrimidine tract binding proteins, PTBP1 and PTBP2

Keppetipola

Yeom

Hernandez

et al. 2016

RNA

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Most human genes generate multiple protein isoforms through alternative pre-mRNA splicing, but the mechanisms controlling alternative splicing choices by RNA binding proteins are not well understood. These proteins can have multiple paralogs expressed in different cell types and exhibiting different splicing activities on target exons. We examined the paralogous polypyrimidine tract binding proteins PTBP1 and PTBP2 to understand how PTBP1 can exhibit greater splicing repression activity on certain exons. Using both an in vivo coexpression assay and an in vitro splicing assay, we show that PTBP1 is more repressive than PTBP2 per unit protein on a target exon. Constructing chimeras of PTBP1 and 2 to determine amino acid features that contribute to their differential activity, we find that multiple segments of PTBP1 increase the repressive activity of PTBP2. Notably, when either RRM1 of PTBP2 or the linker peptide separating RRM2 and RRM3 are replaced with the equivalent PTBP1 sequences, the resulting chimeras are highly active for splicing repression. These segments are distinct from the known region of interaction for the PTBP1 cofactors Raver1 and Matrin3 in RRM2. We find that RRM2 of PTBP1 also increases the repression activity of an otherwise PTBP2 sequence, and that this is potentially explained by stronger binding by Raver1. These results indicate that multiple features over the length of the two proteins affect their ability to repress an exon.

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Structure-function relationships of the polypyrimidine tract binding protein

Cited by 80 publications

References 97 publications

CmRBP50 Protein Phosphorylation Is Essential for Assembly of a Stable Phloem-mobile High-affinity Ribonucleoprotein Complex

CmRBP50 Protein Phosphorylation Is Essential for Assembly of a Stable Phloem-mobile High-affinity Ribonucleoprotein Complex

Activation of picornaviral IRESs by PTB shows differential dependence on each PTB RNA-binding domain

Multiple determinants of splicing repression activity in the polypyrimidine tract binding proteins, PTBP1 and PTBP2

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