Fig 1.
UPF1 organization is very similar in Ustilago maydis and Homo sapiens.
A) Schematic representation of the domain arrangement for UPF1 in Ustilago maydis (umUPF1) and Homo sapiens (hUPF1). The CH domain (green) is responsible for the interaction with UPF2, eRF1 and eRF3. The helicase region contains two RecA domains (yellow). Additional regulatory domains include domain 1B (orange) and domain 1C (red). The amino acid position is shown for the beginning and the end of each peptide. B) The relative position for each domain in both H. sapiens and U. maydis is presented with a summary of the main features reported for the human factor. Positions for the human factor correspond to isoform 1 (Q92900-1).
Fig 2.
Sequence conservation of UPF1 between H. sapiens and U. maydis.
Alignment of the full amino acid sequences for umUPF1 and hUPF1 where the conserved residues are indicated in blue. Each domain is illustrated on top of the sequence using the same color code as in Fig 1. Secondary structural elements are also depicted: rectangles represent α-helices and arrows correspond to β-sheets. Conserved helicase motifs (I, II, II, IV, V and VI) are shown as gray boxes. The loop 349–355 is highlighted in brown, which is interrupted in isoform 1 due to an intronic sequence. The glycine/serine-rich motif corresponds to the dark blue box.
Fig 3.
Post-translational modifications predicted for umUPF1.
The relative position for the different amino acids that could be modified in umUPF1 is shown on top. The table at the bottom includes the sequence bearing either a phosphorylation (p) or ubiquitination (ub) site in H. sapiens and U. maydis. (*) indicates that the modification has been experimentally validated. (°) illustrates the positions where a Threonine in H. sapiens is equivalent to a Serine in U. maydis. HTP corresponds to the number of records in which this modification site was assigned using only proteomic discovery-mode mass spectrometry.
Fig 4.
Evolutionary relationship and domain structure of UPF1 in different species.
The analysis involved 32 amino acidic sequences from animals, plants, fungi and protists. On the left side, the evolutionary history for UPF1 inferred using the UPGMA method is shown. The tree is drawn to scale and the evolutionary analysis was conducted using CLC sequence viewer. Domain architectures and protein size according to InterPro are indicated at right for each species. Five different predicted domains were found, showing different arrangements among the 32 species. The main domain organization includes the UPF2-interacting domain which lies at the N-terminal region (IPR018999, light green) while the P-loop NTPase fold (IPR027417) was found towards the C-terminus in all species except for D. purpureum.
Table 1.
Putative NMD factors identified in Ustilago maydis.
Fig 5.
UPF1 interaction with different proteins.
A) Known protein interactions for hUPF1 using STRING. B) Predicted interactions for umUPF1. In both representations, blue lines join the two proteins involved in the interaction. A solid line indicates a more reliable interaction. The score determined by STRING is also provided for each interaction. C) Domain structural analysis for UPF2, UPF3, and SMG1 (mTOR) in U. maydis and H. sapiens. Domain architectures according to InterPro and protein size are indicated. In UPF2 the MIF4G-like and type 3 domains (IPR016021) are indicated in green and brown respectively and in lila the Up-frameshift suppressor 2 domain (IPR007193). For UPF3 the Nucleotide-binding alpha-beta plait domain (IPR012677) is shown in pink and the Regulator of nonsense-mediated decay UPF3 domain (IPR005120) in grey. For SMG1 and mTOR the important folds are Phosphatidylinositol 3-/4-kinase, the catalytic domain (IPR000403) in orange, an Armadillo-type fold (IPR016024) in Brown, the PIK-related kinase domain (IPR014009) in purple and in lila the FATC domain (IPR003152).
Fig 6.
The tightened conformation of umUPF1 is maintained by the interaction between the CH and RecA2 domains.
On the left, the model obtained for umUPF1 structure was generated using the crystal 2XZL as template. Coloring is as in Fig 1. The detail of the interaction is exhibited in the zoom at right. The key amino acids responsible for this cooperation are shown in pink.
Fig 7.
The CH domain of umUPF1 mediates the interaction with UPF2.
The crystal 2IYK was used as template and the interacting factor corresponds to the human UPF2. The crystal and the model constructed were superposed and only the model for U. maydis is depicted. A hydrophobic surface is involved in the interaction of umUPF1 with the αA-helix in UPF2. Other residues conform a hydrophobic cavity where the βA of UPF2 docks. Key residues in umUPF1 (orange) and the interacting amino acids in UPF2 (yellow) are depicted. Bottom: sequence alignment for the CH domain in all the species used to construct the tree shown in Fig 4. All the amino acids involved in the two hydrophobic regions are highly conserved among species (*).
Fig 8.
In silico disruption of the CH-UPF2 association.
Top panel: original conformation that elicits the hydrophobic interactions of the CH domain in umUPF1 either with the βA (left) or the αA helix (right) of UPF2. Lower panel: in silico mutations for both hydrophobic regions. Key amino acids identified in the CH domain of umUPF1 (orange) and mutated residues (red) are shown.
Fig 9.
Conformational arrangement of umUPF1 in the presence of ATP and ADP.
The models for umUPF1 binding ATP or ADP were constructed using the crystals 2GJK and 2GK6 as template, respectively. A) At the center of the pocket formed by the RecA domains (yellow), all the amino acids that could elicit the interaction with the nucleotide are shown. The residues in the model that could bind ATP are illustrated using strong colors and with light colors those that could connect ADP. B) The ATP binding domain identified for the SF1/2 helicases is depicted. C) An enlarged representation of the ATP binding pocket is presented, showing the identity of the residues involved. D) All the residues involved in the structural arrangement were mutated by Ala.
Fig 10.
RNA binding interaction of umUPF1.
A channel is formed in the umUPF1 molecule eliciting the interaction between RNA and the protein. The residues that form the channel are presented in blue. The RNA molecule appears in black sticks. At the bottom, the detail of this interaction is presented. The identity and position of the key amino acids of umUPF1 involved in the interaction with RNA are depicted. Some residues have been previously reported as necessary for the interaction, while some others are suggested from our observations. This umUPF1 model was constructed using the 2XZO structure as a template.
Fig 11.
Molecular Dynamics of the UPF proteins from H. sapiens and U. maydis.
A) Graphic representation of the RMS calculated for the superposed structures of hUPF1 and umUPF1. A structural alignment was generated for the hUPF1 crystal 2IYK and the model generated for umUPF1. Each amino acid of the umUPF1 protein is colored accordingly to its RMS backbone deviation from the corresponding residue in hUPF1. The scale goes from dark blue for good superposition to red where superposition is poor (Swissmodel). B) Radius of gyration (Rg). Black line corresponds to U. maydis and red line for H. sapiens during the simulation of 10ns at 300K. C) RMSD plot. Root mean square deviation of backbone atoms shown as a function of time for the structure reported for hUPF1 (red, PDB ID: 2iyk) and the modeled structure for umUPF1 (black) during the simulation of 10ns at 300K. D) RMSF plot. Root mean square fluctuations of carbon alpha residues were calculated over time during 10ns simulation at 300K for both hUPF1 (red) and umUPF1 (black).