Lidia Ruiz

Structure of the dimeric exonuclease TREX1 in complex with DNA displays a proline-rich binding site for WW Domains.

TREX1 is the most abundant mammalian 3′ → 5′ DNA exonuclease. It has been described to form part of the SET complex and is responsible for the Aicardi-Goutières syndrome in humans. Here we show that the exonuclease activity is correlated to the binding preferences toward certain DNA sequences. In particular, we have found three motifs that are selected, GAG, ACA, and CTGC. To elucidate how the discrimination occurs, we determined the crystal structures of two murine TREX1 complexes, with a nucleotide product of the exonuclease reaction, and with a single-stranded DNA substrate. Using confocal microscopy, we observed TREX1 both in nuclear and cytoplasmic subcellular compartments. Remarkably, the presence of TREX1 in the nucleus requires the loss of a C-terminal segment, which we named leucine-rich repeat 3. Furthermore, we detected the presence of a conserved proline-rich region on the surface of TREX1. This observation points to interactions with proline-binding domains. The potential interacting motif “PPPVPRPP” does not contain aromatic residues and thus resembles other sequences that select SH3 and/or Group 2 WW domains. By means of nuclear magnetic resonance titration experiments, we show that, indeed, a polyproline peptide derived from the murine TREX1 sequence interacted with the WW2 domain of the elongation transcription factor CA150. Co-immunoprecipitation studies confirmed this interaction with the full-length TREX1 protein, thereby suggesting that TREX1 participates in more functional complexes than previously thought.

The Structure of Prp40 FF1 Domain and Its Interaction with the crn-TPR1 Motif of Clf1 Gives a New Insight into the Binding Mode of FF Domains

The yeast splicing factor Prp40 (pre-mRNA processing protein 40) consists of a pair of WW domains followed by several FF domains. The region comprising the FF domains has been shown to associate with the 5′ end of U1 small nuclear RNA and to interact directly with two proteins, the Clf1 (Crooked neck-like factor 1) and the phosphorylated repeats of the C-terminal domain of RNA polymerase II (CTD-RNAPII). In this work we reported the solution structure of the first FF domain of Prp40 and the identification of a novel ligand-binding site in FF domains. By using chemical shift assays, we found a binding site for the N-terminal crooked neck tetratricopeptide repeat of Clf1 that is distinct and structurally separate from the previously identified CTD-RNAPII binding pocket of the FBP11 (formin-binding protein 11) FF1 domain. No interaction, however, was observed between the Prp40 FF1 domain and three different peptides derived from the CTD-RNAPII protein. Indeed, the equivalent CTD-RNAPII-binding site in the Prp40 FF1 domain is predominantly negatively charged and thus unfavorable for an interaction with phosphorylated peptide sequences. Sequence alignments and phylogenetic tree reconstructions using the FF domains of three functionally related proteins, Prp40, FBP11, and CA150, revealed that Prp40 and FBP11 are not orthologous proteins and supported the different ligand specificities shown by their respective FF1 domains. Our results also revealed that not all FF domains in Prp40 are functionally equivalent. We proposed that at least two different interaction surfaces exist in FF domains that have evolved to recognize distinct binding motifs.

The FF4 and FF5 Domains of Transcription Elongation Regulator 1 (TCERG1) Target Proteins to the Periphery of Speckles

Background: Coordinated transcription and splicing occurs at the periphery of speckles. Results: The FF4 and FF5 domains of transcription elongation regulator 1 (TCERG1) form a structural unit that directs proteins to the periphery of speckles. Conclusion: The FF4 and FF5 domains constitute a novel speckle periphery-targeting signal. Significance: This speckle periphery-targeting signal might participate in the coordination of transcription and splicing. Transcription elongation regulator 1 (TCERG1) is a human factor implicated in interactions with the spliceosome as a coupler of transcription and splicing. The protein is highly concentrated at the interface between speckles (the compartments enriched in splicing factors) and nearby transcription sites. Here, we identified the FF4 and FF5 domains of TCERG1 as the amino acid sequences required to direct this protein to the periphery of nuclear speckles, where coordinated transcription/RNA processing events occur. Consistent with our localization data, we observed that the FF4 and FF5 pair is required to fold in solution, thus suggesting that the pair forms a functional unit. When added to heterologous proteins, the FF4-FF5 pair is capable of targeting the resulting fusion protein to speckles. This represents, to our knowledge, the first description of a targeting signal for the localization of proteins to sites peripheral to speckled domains. Moreover, this “speckle periphery-targeting signal” contributes to the regulation of alternative splicing decisions of a reporter pre-mRNA in vivo.

Solution structure of the fourth FF domain of yeast Prp40 splicing factor.

Prp40 protein was originally identified as a suppressor of 50 end U1 RNA point mutations.1 Prp40 is a U1 snRNP-associated protein that participates in the early steps of yeast premessenger RNA splicing. Prp40 associates with the branch-binding point protein to bring the 50 splicing site and the intron branch point into spatial proximity.2 Additionally, Prp40 has been implicated in the binding to the phosphorylated C-terminal domain (herein referred as phospho-CTD) of RNA polymerase II through regions involving the WW and FF domains.3 However, a subsequent study on the structure of the Prp40 WW domain pair also showed that, in the absence of additional FF domains, the WW domains do not interact with the phospho-CTD repeats.4 The solution structure of the first FF domain of Prp40 has been determined.5 That study also examined the binding of Prp40FF1 to the splicing factor Clf1 and to a pair of bisphospho-CTD repeats. The binding site for the association with the first TPR motif of Clf1 involves the helices a2, the 310, and the N-terminal half of a3. In contrast, no interaction was detected for the Prp40FF1 domain with the phospho-CTD repeats and for the Prp40FF4 domain with the TPR motif of Clf1.5 Recently, other ligand partners have been identified for Prp40 FF domains, namely Snu71, a component of U1 snRNP, and Luc7, a splicing factor associated with U1 snRNP involved in the 50 splicing site recognition.6–8 Furthermore, it was shown that only the region comprising the first two FF domains of Prp40 is critical for yeast viability, whereas the deletion of the region including FF3 and FF4 results in a slow-growth phenotype.6 These results are in agreement with a previous report that showed that a deletion of the FF1 domain and a fragment of FF2 of Prp40 caused yeast lethality.9 To further study the remaining FF domains present in Prp40, we cloned and produced constructs corresponding to the FF2, FF3, and FF4 domains. Of these, only the constructs corresponding to FF4 gave a folded and stable sample. Indeed, several constructs spanning FF2 and FF3 and even that of the FF1-2 pair were either partially structured or unstable after refolding from inclusion bodies. Here we report the solution structure of the Prp40FF4 domain. Furthermore, prompted by the observation that the charge distribution of the FBP11FF1 region involved in the interaction with the bisphospho-CTD repeats10 is partially conserved in Prp40FF4, we also examined whether this domain also interacted with the phosphoCTD repeats, but no binding was detected under our experimental conditions.

RNA recognition and self-association of CPEB4 is mediated by its tandem RRM domains

Cytoplasmic polyadenylation is regulated by the interaction of the cytoplasmic polyadenylation element binding proteins (CPEB) with cytoplasmic polyadenylation element (CPE) containing mRNAs. The CPEB family comprises four paralogs, CPEB1–4, each composed of a variable N-terminal region, two RNA recognition motif (RRM) and a C-terminal ZZ-domain. We have characterized the RRM domains of CPEB4 and their binding properties using a combination of biochemical, biophysical and NMR techniques. Isothermal titration calorimetry, NMR and electrophoretic mobility shift assay experiments demonstrate that both the RRM domains are required for an optimal CPE interaction and the presence of either one or two adenosines in the two most commonly used consensus CPE motifs has little effect on the affinity of the interaction. Both the single RRM1 and the tandem RRM1–RRM2 have the ability to dimerize, although representing a minor population. Self-association does not affect the proteins’ ability to interact with RNA as demonstrated by ion mobility–mass spectrometry. Chemical shift effects measured by NMR of the apo forms of the RRM1–RRM2 samples indicate that the two domains are orientated toward each other. NMR titration experiments show that residues on the β-sheet surface on RRM1 and at the C-terminus of RRM2 are affected upon RNA binding. We propose a model of the CPEB4 RRM1–RRM2–CPE complex that illustrates the experimental data.

NMR Structural Studies on Human p190-A RhoGAPFF1 Revealed that Domain Phosphorylation by the PDGF-Receptor α Requires Its Previous Unfolding

p190-A and -B Rho GAPs (guanosine triphosphatase activating proteins) are the only cytoplasmatic proteins containing FF domains. In p190-A Rho GAP, the region containing the FF domains has been implicated in binding to the transcription factor TFII-I. Moreover, phosphorylation of Tyr308 within the first FF domain inhibits this interaction. Because the structural determinants governing this mechanism remain unknown, we sought to solve the structure of the first FF domain of p190-A Rho GAP (RhoGAPFF1) and to study the potential impact of phosphorylation on the structure. We found that RhoGAPFF1 does not fold with the typical (alpha1-alpha2-3(10)-alpha 3) arrangement of other FF domains. Instead, the NMR data obtained at 285 K show an alpha1-alpha2-alpha 3-alpha 4 topology. In addition, we observed that specific contacts between residues in the first loop and the fourth helix are indispensable for the correct folding and stability of this domain. The structure also revealed that Tyr308 contributes to the domain hydrophobic core. Furthermore, the residues that compose the target motif of the platelet-derived growth factor receptor alpha kinase form part of the alpha 3 helix. We observed that the phosphorylation reaction requires a previous step including domain unfolding, a process that occurs at 310 K. In the absence of phosphorylation, the temperature-dependent RhoGAPFF1 folding/unfolding process is reversible. However, phosphorylation causes an irreversible destabilization of the RhoGAPFF1 structure, which probably accounts for the inhibitory effect that it exerts on the TFII-I interaction. Our results link the ability of a protein domain to be phosphorylated with conformational changes in its three-dimensional structure.

Structural basis for genome wide recognition of 5-bp GC motifs by SMAD transcription factors.

Smad transcription factors activated by TGF-β or by BMP receptors form trimeric complexes with Smad4 to target specific genes for cell fate regulation. The CAGAC motif has been considered as the main binding element for Smad2/3/4, whereas Smad1/5/8 have been thought to preferentially bind GC-rich elements. However, chromatin immunoprecipitation analysis in embryonic stem cells showed extensive binding of Smad2/3/4 to GC-rich cis-regulatory elements. Here, we present the structural basis for specific binding of Smad3 and Smad4 to GC-rich motifs in the goosecoid promoter, a nodal-regulated differentiation gene. The structures revealed a 5-bp consensus sequence GGC(GC)|(CG) as the binding site for both TGF-β and BMP-activated Smads and for Smad4. These 5GC motifs are highly represented as clusters in Smad-bound regions genome-wide. Our results provide a basis for understanding the functional adaptability of Smads in different cellular contexts, and their dependence on lineage-determining transcription factors to target specific genes in TGF-β and BMP pathways.Smad transcription factors are part of the TGF-β signal transduction pathways and are recruited to the genome by cell lineage-defining factors. Here, the authors identify specific Smad binding GC-rich motifs and provide structural information showing Smad3 and Smad4 bound to these motifs.

TGIF1 homeodomain interacts with Smad MH1 domain and represses TGF-β signaling

Abstract TGIF1 is a multifunctional protein that represses TGF-β-activated transcription by interacting with Smad2-Smad4 complexes. We found that the complex structure of TGIF1–HD bound to the TGACA motif revealed a combined binding mode that involves the HD core and the major groove, on the one hand, and the amino-terminal (N-term) arm and the minor groove of the DNA, on the other. We also show that TGIF1–HD interacts with the MH1 domain of Smad proteins, thereby indicating that TGIF1–HD is also a protein-binding domain. Moreover, the formation of the HD-MH1 complex partially hinders the DNA-binding site of the complex, preventing the efficient interaction of TGIF1–HD with DNA. We propose that the binding of the TGIF1 C-term to the Smad2-MH2 domain brings both the HD and MH1 domain into close proximity. This local proximity facilitates the interaction of these DNA-binding domains, thus strengthening the formation of the protein complex versus DNA binding. Once the protein complex has been formed, the TGIF1-Smad system would be released from promoters/enhancers, thereby illustrating one of the mechanisms used by TGIF1 to exert its function as an active repressor of Smad-induced TGF-β signaling.