José Manuel Pérez-Cañadillas

The C-terminal Domains of Vertebrate CstF-64 and Its Yeast Orthologue Rna15 Form a New Structure Critical for mRNA 3′-End Processing

Yeast Rna15 and its vertebrate orthologue CstF-64 play critical roles in mRNA 3 ′-end processing and in transcription termination downstream of poly(A) sites. These proteins contain N-terminal domains that recognize the poly(A) site, but little is known about their highly conserved C-terminal regions. Here we show by NMR that the C-terminal domains of CstF-64 and Rna15 fold into a three-helix bundle with an uncommon topological arrangement. The structure defines a cluster of evolutionary conserved yet exposed residues we show to be essential for the interaction between Pcf11 and Rna15. Furthermore, we demonstrate that this interaction is critical for the function of Rna15 in 3 ′-end processing but dispensable for transcription termination. The C-terminal domain of the Rna15 homologue Pti1 contains critical sequence alterations within this region that are predicted to prevent Pcf11 interaction, providing an explanation for the distinct functions of these two closely related proteins in the 3 ′-end formation of RNA polymerase II transcripts. These results define the role of the C-terminal half of Rna15 and provide insight into the network of protein/protein interactions responsible for assembly of the 3 ′-end processing apparatus.

Role of histidine‐50, glutamic acid‐96, and histidine‐137 in the ribonucleolytic mechanism of the ribotoxin α‐sarcin

α‐Sarcin is a ribotoxin secreted by the mold Aspergillus giganteus that degrades the ribosomal RNA by acting as a cyclizing ribonuclease. Three residues potentially involved in the mechanism of catalysis—histidine‐50, glutamic acid‐96, and histidine‐137—were changed to glutamine. Three dif‐ ferent single mutation variants (H50Q, E96Q, H137Q) as well as a double variant (H50/137Q) and a triple variant (H50/137Q/E96Q) were prepared and isolated to homogeneity. These variants were spectroscopically (circular dichroism, fluorescence emission, and proton nuclear magnetic resonance) characterized. According to these results, the three‐dimensional structure of these variants of α‐sarcin was preserved; only very minor local changes were detected. All the variants were inactive when assayed against either intact ribosomes or poly(A). The effect of pH on the ribonucleolytic activity of α‐sarcin was evaluated against the ApA dinucleotide. This assay revealed that only the H50Q variant still retained its ability to cleave a phosphodiester bond, but it did so to a lesser extent than did wild‐type α‐sarcin. The results obtained are interpreted in terms of His137 and Glu96 as essential residues for the catalytic activity of α‐sarcin (His137 as the general acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by α‐sarcin. Proteins 1999;37:474–484. ©1999 Wiley‐Liss, Inc.

Solution structure and dynamics of ribonuclease Sa.

We have used NMR methods to characterize the structure and dynamics of ribonuclease Sa in solution. The solution structure of RNase Sa was obtained using the distance constraints provided by 2,276 NOEs and the C6C96 disulfide bond. The 40 resulting structures are well determined; their mean pairwise RMSD is 0.76 Å (backbone) and 1.26 Å (heavy atoms). The solution structures are similar to previously determined crystal structures, especially in the secondary structure, but exhibit new features: the loop composed of Pro 45 to Ser 48 adopts distinct conformations and the rings of tyrosines 51, 52, and 55 have reduced flipping rates. Amide protons with greatly reduced exchange rates are found predominantly in interior β‐strands and the α‐helix, but also in the external 3/10 helix and edge β‐strand linked by the disulfide bond. Analysis of 15N relaxation experiments (R1, R2, and NOE) at 600 MHz revealed five segments, consisting of residues 1–5, 28–31, 46–50, 60–65, 74–77, retaining flexibility in solution. The change in conformation entropy for RNase SA folding is smaller than previously believed, since the native protein is more flexible in solution than in a crystal. Proteins 2001;44:200–211.

Leucine 145 of the ribotoxin α-sarcin plays a key role for determining the specificity of the ribosome-inactivating activity of the protein

Secreted fungal RNases, represented by RNase T1, constitute a family of structurally related proteins that includes ribotoxins such as α‐sarcin. The active site residues of RNase T1 are conserved in all fungal RNases, except for Phe 100 that is not present in the ribotoxins, in which Leu 145 occupies the equivalent position. The mutant Leu145Phe of α‐sarcin has been recombinantly produced and characterized by spectroscopic methods (circular dichroism, fluorescence spectroscopy, and NMR). These analyses have revealed that the mutant protein retained the overall conformation of the wild‐type α‐sarcin. According to the analyses performed, Leu 145 was shown to be essential to preserve the electrostatic environment of the active site that is required to maintain the anomalous low pKa value reported for the catalytic His 137 of α‐sarcin. Enzymatic characterization of the mutant protein has revealed that Leu 145 is crucial for the specific activity of α‐sarcin on ribosomes.

Two Singular Types of Ccch Tandem Zinc Finger in Nab2P Contribute to Polyadenosine RNA Recognition.

The seven C-terminal CCCH-type zinc fingers of Nab2p bind the poly(A) tail of mRNA (∼A25). Using NMR, we demonstrated that the first four (Zf1-Zf4) contain two structurally independent tandems (TZF12 and TZF34) and bind A12 with moderate affinity (KD = 2.3 μM). Nab2p TZF12 contains a long α helix that contacts the zinc fingers Zf1 and Zf2 to arrange them similarly to Zf6-7 in the Nab2p Zf5-7 structure. Nab2p TZF34 exhibits a distinctive two-fold symmetry of the zinc centers with mutual recognition of histidine ligands. Our mutagenesis and NMR data demonstrate that the α helix of TZF12 and Zf3 of TZF34 define the RNA-binding interface, while Zf1, Zf2, and Zf4 seem to be excluded. These results further our understanding of polyadenosine RNA recognition by the CCCH domain of Nab2p. Moreover, we describe a hypothetical mechanism for controlling poly(A) tail length with specific roles for TZF12, TZF34, and Zf5-7 domains.

NMR structure note: PHD domain from death inducer obliterator protein and its interaction with H3K4me3

The plant homeodomain (PHD) modules are small 50–80 amino acid zinc fingers present in many nuclear proteins, which recognise histone post-translational modifications, i.e. lysine methylation and acetylation (Li and Li 2012; Musselman et al. 2012; Sanchez and Zhou 2011; Baker et al. 2008). These modifications play an essential role in the regulation of transcription, activation or repression depending on the nature and extent of the modification and on the target lysine. Misreading of these epigenetic marks has been related with many human pathological states, such as cancer, immunological and neurological diseases (Musselman et al. 2012; Baker et al. 2008). The death inducer obliterator (Dido) gene encodes three protein isoforms of different lengths. The longest and most broadly expressed, Dido3, is a nuclear protein that associates to the spindle pole in mitosis and to the synaptonemal complex in meiosis. Alterations in the expression of the Dido gene have been related to myeloid neoplasms in humans (Futterer et al. 2005). Based on pull-down assays, the N-terminal region of murine Dido3 has been reported to associate to histone H3 (Prieto et al. 2009). Histone recognition requires the PHD motif present in all Dido isoforms at their common N-terminal region (Fig. 1a; Prieto et al. 2009). It is noticeable that the PHD domain sequence in Dido genes from different organisms is completely conserved, whilst the overall identities lie in the range 60–96 %. Surface plasmon resonance experiments indicated that, in vitro, the Dido PHD domain is able to bind histone H3-derived peptides. The affinity is higher for the peptide with trimethylated-lysine 4 (H3K4me3) than for its non-methylated counterpart. Dido PHD domain was shown to recognise H3K4me3 also in vivo, and the methylation state of lysine 4 seems to be involved in the cellular localization of Dido3 (Prieto et al. 2009). Thus, knowledge of the molecular basis for the interaction between the PHD domain of Dido and histone H3K4me3 would improve current understanding of the biological roles played by Dido. With this aim in mind we proceeded to determine the structure of the PHD domain of Dido (DidoPHD), residues 265–322 in humans (Fig. 1a), and to map its interaction with a 12-residue H3K4me3 histone peptide (Fig. 1b).

Pub1p C-Terminal RRM Domain Interacts with Tif4631p through a Conserved Region Neighbouring the Pab1p Binding Site

Pub1p, a highly abundant poly(A)+ mRNA binding protein in Saccharomyces cerevisiae, influences the stability and translational control of many cellular transcripts, particularly under some types of environmental stresses. We have studied the structure, RNA and protein recognition modes of different Pub1p constructs by NMR spectroscopy. The structure of the C-terminal RRM domain (RRM3) shows a non-canonical N-terminal helix that packs against the canonical RRM fold in an original fashion. This structural trait is conserved in Pub1p metazoan homologues, the TIA-1 family, defining a new class of RRM-type domains that we propose to name TRRM (TIA-1 C-terminal domain-like RRM). Pub1p TRRM and the N-terminal RRM1-RRM2 tandem bind RNA with high selectivity for U-rich sequences, with TRRM showing additional preference for UA-rich ones. RNA-mediated chemical shift changes map to β-sheet and protein loops in the three RRMs. Additionally, NMR titration and biochemical in vitro cross-linking experiments determined that Pub1p TRRM interacts specifically with the N-terminal region (1–402) of yeast eIF4G1 (Tif4631p), very likely through the conserved Box1, a short sequence motif neighbouring the Pab1p binding site in Tif4631p. The interaction involves conserved residues of Pub1p TRRM, which define a protein interface that mirrors the Pab1p-Tif4631p binding mode. Neither protein nor RNA recognition involves the novel N-terminal helix, whose functional role remains unclear. By integrating these new results with the current knowledge about Pub1p, we proposed different mechanisms of Pub1p recruitment to the mRNPs and Pub1p-mediated mRNA stabilization in which the Pub1p/Tif4631p interaction would play an important role.

Gbp2 interacts with THO/TREX through a novel type of RRM domain

Metazoan SR and SR-like proteins are important regulatory factors in RNA splicing, export, translation and RNA decay. We determined the NMR structures and nucleic acid interaction modes of Gbp2 and Hrb1, two paralogous budding yeast proteins with similarities to mammalian SR proteins. Gbp2 RRM1 and RRM2 recognise preferentially RNAs containing the core motif GGUG. Sequence selectivity resides in a non-canonical interface in RRM2 that is highly related to the SRSF1 pseudoRRM. The atypical Gbp2/Hrb1 C-terminal RRM domains (RRM3) do not interact with RNA/DNA, likely because of their novel N-terminal extensions that block the canonical RNA binding interface. Instead, we discovered that RRM3 is crucial for interaction with the THO/TREX complex and identified key residues essential for this interaction. Moreover, Gbp2 interacts genetically with Tho2 as the double deletion shows a synthetic phenotype and preventing Gbp2 interaction with the THO/TREX complex partly supresses gene expression defect associated with inactivation of the latter complex. These findings provide structural and functional insights into the contribution of SR-like proteins in the post-transcriptional control of gene expression.

The structure of transcription termination factor Nrd1 reveals an original mode for GUAA recognition

Abstract Transcription termination of non-coding RNAs is regulated in yeast by a complex of three RNA binding proteins: Nrd1, Nab3 and Sen1. Nrd1 is central in this process by interacting with Rbp1 of RNA polymerase II, Trf4 of TRAMP and GUAA/G terminator sequences. We lack structural data for the last of these binding events. We determined the structures of Nrd1 RNA binding domain and its complexes with three GUAA-containing RNAs, characterized RNA binding energetics and tested rationally designed mutants in vivo. The Nrd1 structure shows an RRM domain fused with a second α/β domain that we name split domain (SD), because it is formed by two non-consecutive segments at each side of the RRM. The GUAA interacts with both domains and with a pocket of water molecules, trapped between the two stacking adenines and the SD. Comprehensive binding studies demonstrate for the first time that Nrd1 has a slight preference for GUAA over GUAG and genetic and functional studies suggest that Nrd1 RNA binding domain might play further roles in non-coding RNAs transcription termination.