Tudor-like Domain Research Articles

Molecular Basis for SPINDOC-Spindlin1 Engagement and Its Role in Transcriptional Attenuation

Spindlin1 is a histone reader with three Tudor-like domains and its transcriptional co-activator activity could be attenuated by SPINDOC. The first two Tudors are involved in histone methylation readout, while the function of Tudor 3 is largely unknown. Here our structural and binding studies revealed an engagement mode of SPINDOC-Spindlin1, in which a hydrophobic motif of SPINDOC, DOCpep3, stably interacts with Spindlin1 Tudor 3, and two neighboring K/R-rich motifs, DOCpep1 and DOCpep2, bind to the acidic surface of Spindlin1 Tudor 2. Although DOCpep3-Spindlin1 engagement is compatible with histone readout, an extended SPINDOC fragment containing the K/R-rich region attenuates histone or TCF4 binding by Spindlin1 due to introduced competition. This inhibitory effect is more pronounced for weaker binding targets but not for strong ones such as H3 “K4me3-K9me3” bivalent mark. Further ChIP-seq and RT-qPCR indicated that SPINDOC could promote genomic relocation of Spindlin1, thus modulate downstream gene transcription. Collectively, we revealed multivalent engagement between SPINDOC and Spindlin1, in which a hydrophobic motif acts as the primary binding site for stable SPINDOC-Spindlin1 association, while K/R-rich region modulates the target selectivity of Spindlin1 via competitive inhibition, therefore attenuating the transcriptional co-activator activity of Spindlin1.

Searching for Biological Function of the Mysterious PA2504 Protein from Pseudomonas aeruginosa.

For nearly half of the proteome of an important pathogen, Pseudomonas aeruginosa, the function has not yet been recognised. Here, we characterise one such mysterious protein PA2504, originally isolated by us as a sole partner of the RppH RNA hydrolase involved in transcription regulation of multiple genes. This study aims at elucidating details of PA2504 function and discussing its implications for bacterial biology. We show that PA2504 forms homodimers and is evenly distributed in the cytoplasm of bacterial cells. Molecular modelling identified the presence of a Tudor-like domain in PA2504. Transcriptomic analysis of a ΔPA2504 mutant showed that 42 transcripts, mainly coding for proteins involved in sulphur metabolism, were affected by the lack of PA2504. In vivo crosslinking of cellular proteins in the exponential and stationary phase of growth revealed several polypeptides that bound to PA2504 exclusively in the stationary phase. Mass spectrometry analysis identified them as the 30S ribosomal protein S4, the translation elongation factor TufA, and the global response regulator GacA. These results indicate that PA2504 may function as a tether for several important cellular factors.

Spindlin1 alters the metaphase to anaphase transition in meiosis I through regulation of BUB3 expression in porcine oocytes.

Spindlin 1 (SPIN1), which contains Tudor-like domains, regulates maternal transcripts via interaction with a messenger RNA (mRNA)-binding protein. SPIN1 is involved in tumorigenesis in somatic cells and is highly expressed in cancer cells. Nevertheless, the role of SPIN1 in porcine oocyte maturation remains totally unknown. To explore the function of SPIN1 in porcine oocyte maturation, knockdown, and overexpression techniques were used. SPIN1 mRNA was identified in maternal stages ranging from GV to MII. SPIN1 was localized in the cytoplasm and to chromosomes during meiosis. SPIN1 knockdown accelerated first polar body extrusion. Oocytes with overexpressed SPIN1 were arrested at the MI stage. SPIN1 depletion caused meiotic spindle defects and chromosome instability. The BUB3 signal was investigated, confirming that SPIN1 affects the stability of Bub3 mRNA as well as BUB3 expression. Further, overexpression of SPIN1 inhibited the degradation and regulation of G2/mitotic-specific cyclin-B1. In summation, SPIN1 regulates the meiotic cell cycle by modulating the activation of the spindle assembly checkpoint.

Are Cajal Bodies Droplet Organelles?

Cajal Bodies(CBs) are subnuclear organelles responsible for spliceosomal snRNP maturation in higher eukaryotes. Vertebrates must maintain high snRNP levels during development, and a loss of CBs results in embryonic arrest due to defects in splicing. Like nucleoli and other cellular bodies, CBs are not delimited by lipid bilayers and are RNA-rich. Current models for the formation of such “droplet organelles” invoke phase transitions promoted by intrinsically disordered regions (IDRs) of key components. CBs are scaffolded by the protein coilin, which is assumed to be largely disordered; however, coilin's biochemical and biophysical properties have not been fully examined. It is currently unclear which coilin regions are structured and whether coilin itself mediates a phase transition. For example, a protein or RNA interaction partner may do so. We have purified coilin and subjected it to circular dichroism spectroscopy, finding that at least portions of coilin can adopt an ordered state. In vivo staining with amyloid sensitive fluorescent dyes indicates that this structure is not cross-beta-sheet. Protein folds predicted with RaptorX include: 1) a ubiquitin-like fold at the N-terminus followed by 2) an IDR and 3) a C-terminal tudor-like domain. Prior immunoprecipitation results have shown that the N-terminal domain mediates direct or indirect coilin “self-interaction”, and UV crosslinking revealed RNA binding properties. We have identified single amino acid mutations that disrupt CBs, suggesting specific functional sites within coilin are currently employing a protein-protein crosslinker (DPS) to implicate interactors. The major species appears to consist of coilin-coilin dimers. Thus far, our findings are consistent with a model wherein the IDR promotes a phase transition yet two structured regions impose specificity: an N-terminal coilin-coilin interaction domain and a C-terminal domain that binds to SMN, snRNP proteins and possibly snRNA.

The PipX Protein, When Not Bound to Its Targets, Has Its Signaling C-Terminal Helix in a Flexed Conformation.

PipX, an 89-residue protein, acts as a coactivator of the global nitrogen regulator NtcA in cyanobacteria. NtcA-PipX interactions are regulated by 2-oxoglutarate (2-OG), an inverse indicator of the ammonia abundance, and by PII, a protein that binds to PipX at low 2-OG concentrations. The structure of PipX, when bound to NtcA or PII, consists of an N-terminal, five-stranded β-sheet (conforming a Tudor-like domain), and two long α-helices. These helices adopt either a flexed conformation, where they are in close contact and in an antiparallel mutual orientation, also packing against the β-sheet, or an open conformation (observed only in the PII-PipX complex) where the last α-helix moves apart from the rest of the protein. The aim of this work was to study the structure and dynamics of isolated PipX in solution by NMR. The backbone chemical shifts, the hydrogen-exchange, and the NOE patterns indicated that the isolated, monomeric PipX structure was formed by an N-terminal five-stranded β-sheet and two C-terminal α-helices. Furthermore, the observed NOEs between the two helices, and of α-helix2 with β-strand2 suggested that PipX adopted a flexed conformation. The β-strands 1 and 5 were highly flexible, as shown by the lack of interstrand backbone-backbone NOEs; in addition, the 15N-dynamics indicated that the C terminus of β-strand4 and the following β-turn (Phe42-Thr47), and the C-cap of α-helix1 (Arg70-Asn71) were particularly mobile. These two regions could act as hinges, allowing PipX to interact with its partners, including PlmA in the newly recognized PII-PipX-PlmA ternary complex.

Structure-function dissection of Myxococcus xanthus CarD N-terminal domain, a defining member of the CarD_CdnL_TRCF family of RNA polymerase interacting proteins.

Two prototypes of the large CarD_CdnL_TRCF family of bacterial RNA polymerase (RNAP)-binding proteins, Myxococcus xanthus CarD and CdnL, have distinct functions whose molecular basis remain elusive. CarD, a global regulator linked to the action of several extracytoplasmic function (ECF) σ-factors, binds to the RNAP β subunit (RNAP-β) and to protein CarG via an N-terminal domain, CarDNt, and to DNA via an intrinsically unfolded C-terminal domain resembling eukaryotic high-mobility-group A (HMGA) proteins. CdnL, a CarDNt-like protein that is essential for cell viability, is implicated in σA-dependent rRNA promoter activation and interacts with RNAP-β but not with CarG. While the HMGA-like domain of CarD by itself is inactive, we find that CarDNt has low but observable ability to activate ECF σ-dependent promoters in vivo, indicating that the C-terminal DNA-binding domain is required to maximize activity. Our structure-function dissection of CarDNt reveals an N-terminal, five-stranded β -sheet Tudor-like domain, CarD1–72, whose structure and contacts with RNAP-β mimic those of CdnL. Intriguingly, and in marked contrast to CdnL, CarD mutations that disrupt its interaction with RNAP-β did not annul activity. Our data suggest that the CarDNt C-terminal segment, CarD61–179, may be structurally distinct from its CdnL counterpart, and that it houses at least two distinct and crucial function determinants: (a) CarG-binding, which is specific to CarD; and (b) a basic residue stretch, which is also conserved and functionally required in CdnL. This study highlights the evolution of shared and divergent interactions in similar protein modules that enable the distinct activities of two related members of a functionally important and widespread bacterial protein family.

Virus control goes epigenetic.

Cells have developed a number of mechanisms to detect and suppress microbial infections. The use of pattern recognition receptors (PRRs) is one such mechanism. PRRs may be expressed on the cell surface, in the cytosol, or in the endosomes, to detect pathogen-associated molecular patterns (PAMPs) or the danger signals released by damaged cells (i.e., damage-associated molecular patterns; DAMPs). PRR activation triggers a signaling cascade, resulting in the production of antimicrobial cytokines, including type I interferons. These cytokines will in turn induce the expression of genes, such as interferon-stimulated genes (ISGs), that exhibit antimicrobial activities. In addition to PRRs, autophagy is another intracellular defense mechanism. Autophagy is used by the cells to remove intracellular pathogens in a process known as xenophagy [1]. Many viruses have developed different mechanisms to suppress these innate immune responses for their survival. A notable example is the cleavage of the mitochondrial antiviral signaling protein (MAVS) by the hepatitis C virus (HCV) NS3 protease [2]. MAVS is an important downstream adaptor molecule of RIG-I, which is a cytosolic PRR that can be activated by the HCV genomic RNA. Another example is the influenza virus NS1 protein, which binds to the E3 ubiquitin ligase TRIM25 to prevent it from activating RIG-I [3]. Similarly, the autophagy pathway may also be suppressed by viruses or even exploited by viruses to benefit their own replication. The former is typified by the herpes simplex virus-1 (HSV-1) ICP34.5 protein, which binds to Beclin-1 to prevent it from binding to and activating the class III phosphatidylinositol-3-kinase, an enzyme important for the initiation of autophagy. An example of the latter is HCV, which uses autophagy to enhance its replication [4]. In the report by Ducroux et al. [5], the authors presented yet another type of cellular control of viral infections. By using a tandem-affinity purification approach, the authors identified Spindlin1 as a binding partner of the hepatitis B virus (HBV) X protein (HBx). They subsequently discovered that Spindlin1 could suppress the replication of HBV, as its overexpression reduced the levels of HBV RNA and DNA replicative intermediates. On the contrary, depletion of Spindlin1 by RNA interference enhanced HBV replication. They then demonstrated that the suppressive effect of Spindlin1 on HBV was at the transcriptional level, as the overexpression of Spindlin1 reduced the transcriptional activity of HBV DNA by 80% in a nuclear run-on experiment. This effect of Spindlin1 on HBV was specific, as it was not observed with the cellular gene cyclin A2. Spindlin1 contains three Tudor-like domains and can bind to trimethylated lysine 4 (H3K4me3) and asymmetric dimethylated arginine 8 (H3R8me2a) of histone H3 [6]. In the chromatin immunoprecipitation (ChIP) assay, Ducroux et al. demonstrated that Spindlin1 could bind to the HBV covalently closed circular DNA (cccDNA) genome. Interestingly, this binding was enhanced if the expression of HBx from the viral genome was abolished, suggesting an inhibitory role of HBx in the binding of Spindlin1 to the HBV genome. It does not appear likely that Spindlin1 binds to HBV cccDNA via H3K4me3, though, as the depletion of Spindlin1 actually led to a significant increase of the level of H3K4me3 in the HBV cccDNA. Their results suggested that Spindlin1, by binding to the HBV cccDNA, inhibits this post-translational modification of histone H3, which is associated with actively transcribed genes (Figure 1) [7]. Figure 1 Model for epigenetic regulation of HBV gene expression by Spindlin1. A similar suppressive effect of Spindlin1 on the transcription of HSV-1 RNA was also observed by the authors. They found that the depletion of Spindlin1 also increased the ICP27 RNA level of HSV-1. In contrast, Spindlin1 had no effect on the replication of HCV, an RNA virus. Their results raise the possibility that Spindlin1, either by itself or by interacting with an unknown partner or partners, may represent a new class of PRRs that detect and suppress gene expression of DNA viruses by reducing the H3K4me3 level on the viral genome. If Spindlin1 is indeed a new PRR, then it will be interesting to determine how Spindlin1 differentiates episomal viral DNA from self DNA and what PAMPs it recognizes on the viral DNA. In their studies on HSV-1, Ducroux et al. did not demonstrate whether Spindlin1 could indeed reduce the H3K4me3 level associated with the HSV-1 genome, which must be demonstrated if the ability of Spindlin1 to exert epigenetic control on gene expression of DNA viruses is to be generalized. It will also be interesting in the future to determine whether Spindlin1 exhibits the same suppressive effect on other DNA viruses such as papillomaviruses and polyomaviruses and whether these DNA viruses also express gene products that can antagonize the suppressive effect of Spindlin1.

A Tudor Domain Protein SPINDLIN1 Interacts with the mRNA-Binding Protein SERBP1 and Is Involved in Mouse Oocyte Meiotic Resumption

Mammalian oocytes are arrested at prophase I of meiosis, and resume meiosis prior to ovulation. Coordination of meiotic arrest and resumption is partly dependent on the post-transcriptional regulation of maternal transcripts. Here, we report that, SPINDLIN1 (SPIN1), a maternal protein containing Tudor-like domains, interacts with a known mRNA-binding protein SERBP1, and is involved in regulating maternal transcripts to control meiotic resumption. Mouse oocytes deficient for Spin1 undergo normal folliculogenesis, but are defective in resuming meiosis. SPIN1, via its Tudor-like domain, forms a ribonucleoprotein complex with SERBP1, and regulating mRNA stability and/or translation. The mRNA for the cAMP-degrading enzyme, PDE3A, is reduced in Spin1 mutant oocytes, possibly contributing to meiotic arrest. Our study demonstrates that Spin1 regulates maternal transcripts post-transcriptionally and is involved in meiotic resumption.

Distinct mode of methylated lysine-4 of histone H3 recognition by tandem tudor-like domains of Spindlin1

Recognition of methylated histone tail lysine residues by tudor domains plays important roles in epigenetic control of gene expression and DNA damage response. Previous studies revealed the binding of methyllysine in a cage of aromatic residues, but the molecular mechanism by which the sequence specificity for surrounding histone tail residues is achieved remains poorly understood. In the crystal structure of a trimethylated histone H3 lysine 4 (H3K4) peptide bound to the tudor-like domains of Spindlin1 presented here, an atypical mode of methyllysine recognition by an aromatic pocket of Spindlin1 is observed. Furthermore, the histone sequence is recognized in a distinct manner involving the amino terminus and a pair of arginine residues of histone H3, and disruption of the binding impaired stimulation of pre-RNA expression by Spindlin1. Our analysis demonstrates considerable diversities of methyllysine recognition and sequence-specific binding of histone tails by tudor domains, and the revelation furthers the understanding of tudor domain proteins in deciphering epigenetic marks on histone tails.

The Yb Body, a Major Site for Piwi-associated RNA Biogenesis and a Gateway for Piwi Expression and Transport to the Nucleus in Somatic Cells

Despite exciting progress in understanding the Piwi-interacting RNA (piRNA) pathway in the germ line, less is known about this pathway in somatic cells. We showed previously that Piwi, a key component of the piRNA pathway in Drosophila, is regulated in somatic cells by Yb, a novel protein containing an RNA helicase-like motif and a Tudor-like domain. Yb is specifically expressed in gonadal somatic cells and regulates piwi in somatic niche cells to control germ line and somatic stem cell self-renewal. However, the molecular basis of the regulation remains elusive. Here, we report that Yb recruits Armitage (Armi), a putative RNA helicase involved in the piRNA pathway, to the Yb body, a cytoplasmic sphere to which Yb is exclusively localized. Moreover, co-immunoprecipitation experiments show that Yb forms a complex with Armi. In Yb mutants, Armi is dispersed throughout the cytoplasm, and Piwi fails to enter the nucleus and is rarely detectable in the cytoplasm. Furthermore, somatic piRNAs are drastically diminished, and soma-expressing transposons are desilenced. These observations indicate a crucial role of Yb and the Yb body in piRNA biogenesis, possibly by regulating the activity of Armi that controls the entry of Piwi into the nucleus for its function. Finally, we discovered putative endo-siRNAs in the flamenco locus and the Yb dependence of their expression. These observations further implicate a role for Yb in transposon silencing via both the piRNA and endo-siRNA pathways.

The nitrogen interaction network in Synechococcus WH5701, a cyanobacterium with two PipX and two PII-like proteins

Nitrogen regulation involves the formation of different types of protein complexes between signal transducers and their transcriptional or metabolic targets. In oxygenic phototrophs, the signal integrator P(II) activates the enzyme N-acetyl-l-glutamate kinase (NAGK) by complex formation. P(II) also interacts with PipX, a protein with a tudor-like domain that mediates contacts with P(II) and with the transcriptional regulator NtcA, to which it binds to increase its activity. Here, we use a combination of in silico, yeast two-hybrid and in vitro approaches to investigate the nitrogen regulation network of Synechococcus WH5701, a marine cyanobacterium with two P(II) (GlnB_A and GlnB_B) and two PipX (PipX_I and PipX_II) proteins. Our results indicate that GlnB_A is functionally equivalent to the canonical P(II) protein from Synechococcus elongatus. GlnB_A interacted with PipX and NAGK proteins and stimulated NAGK activity, counteracting arginine inhibition. GlnB_B had only a slight stimulatory effect on NAGK activity, but its potential to bind effectors and form heterotrimers in Synechococcus WH5701 indicates additional regulatory functions. PipX_II, and less evidently PipX_I, specifically interacted with GlnB_A and NtcA, supporting a role for both Synechococcus WH5701 PipX proteins in partner swapping with GlnB_A and NtcA.

Structural basis for the regulation of NtcA-dependent transcription by proteins PipX and PII

PII, an ancient and widespread signaling protein, transduces nitrogen/carbon/energy abundance signals through interactions with target proteins. We clarify structurally how PII regulates gene expression mediated by the transcription factor NtcA, the global nitrogen regulator of cyanobacteria, shedding light on NtcA structure and function and on how NtcA is activated by 2-oxoglutarate (2OG) and coactivated by the nonenzymatic PII target, protein PipX. We determine for the cyanobacteria Synechococcus elongatus the crystal structures of the PII-PipX and PipX-NtcA complexes and of NtcA in active and inactive conformations (respective resolutions, 3.2, 2.25, 2.3, and 3.05 A). The structures and the conclusions derived from them are consistent with the results of present and prior site-directed mutagenesis and functional studies. A tudor-like domain (TLD) makes up most of the PipX structure and mediates virtually all the contacts of PipX with PII and NtcA. In the PII-PipX complex, one PII trimer sequesters the TLDs of three PipX molecules between its body and its extended T loops, preventing PipX activation of NtcA. Changes in T loop conformation triggered by 2OG explain PII-PipX dissociation when 2OG is bound. The structure of active dimeric NtcA closely resembles that of the active cAMP receptor protein (CRP). This strongly suggests that with these proteins DNA binding, transcription activation, and allosteric regulation occur by common mechanisms, although the effectors are different. The PipX-NtcA complex consists of one active NtcA dimer and two PipX monomers. PipX coactivates NtcA by stabilizing its active conformation and by possibly helping recruit RNA polymerase but not by providing extra DNA contacts.

Crystal Structure of the C-Terminal Domain of a Flagellar Hook-Capping Protein from Xanthomonas campestris

The crystal structure of the C-terminal domain of a hook-capping protein FlgD from the plant pathogen Xanthomonas campestris (Xc) has been determined to a resolution of ca 2.5 Å using X-ray crystallography. The monomer of whole FlgD comprises 221 amino acids with a molecular mass of 22.7 kDa, but the flexible N-terminus is cleaved for up to 75 residues during crystallization. The final structure of the C-terminal domain reveals a novel hybrid comprising a tudor-like domain interdigitated with a fibronectin type III domain. The C-terminal domain of XcFlgD forms three types of dimers in the crystal. In agreement with this, analytical ultracentrifugation and gel filtration experiments reveal that they form a stable dimer in solution. From these results, we propose that the Xc flagellar hook cap protein FlgD comprises two individual domains, a flexible N-terminal domain that cannot be detected in the current study and a stable C-terminal domain that forms a stable dimer.

Structure of Human Spindlin1: TANDEM TUDOR-LIKE DOMAINS FOR CELL CYCLE REGULATION

Spindlin1, a meiotic spindle-binding protein that is highly expressed in ovarian cancer cells, was first identified as a gene involved in gametogenesis. It appeared to be a target for cell cycle-dependent phosphorylation and was demonstrated to disturb the cell cycle. Here we report the crystal structure of human spindlin1 to 2.2A of resolution, representing the first three-dimensional structure from the spin/ssty (Y-linked spermiogenesis-specific transcript) gene family. The refined structure, containing three repeats of five/four anti-parallel beta-strands, exhibits a novel arrangement of tandem Tudor-like domains. Two phosphate ions, chelated by Thr-95 and other residues, appear to stabilize the long loop between domains I and II, which might mediate the cell cycle regulation activity of spindlin1. Flow cytometry experiments indicate that cells expressing spindlin1 display a different cell cycle distribution in mitosis, whereas those expressing a T95A mutant, which had a great decrease in phosphorous content, have little effect on the cell cycle. We further identified associations of spindlin1 with nucleic acid to provide a biochemical basis for its cell cycle regulation and other functions.

Structural Basis for Bacterial Transcription-Coupled DNA Repair

Coupling of transcription and DNA repair in bacteria is mediated by transcription-repair coupling factor (TRCF, the product of the mfd gene), which removes transcription elongation complexes stalled at DNA lesions and recruits the nucleotide excision repair machinery to the site. Here we describe the 3.2 A-resolution X-ray crystal structure of Escherichia coli TRCF. The structure consists of a compact arrangement of eight domains, including a translocation module similar to the SF2 ATPase RecG, and a region of structural similarity to UvrB. Biochemical and genetic experiments establish that another domain with structural similarity to the Tudor-like domain of the transcription elongation factor NusG plays a critical role in TRCF/RNA polymerase interactions. Comparison with the translocation module of RecG as well as other structural features indicate that TRCF function involves large-scale conformational changes. These data, along with a structural model for the interaction of TRCF with the transcription elongation complex, provide mechanistic insights into TRCF function.

Two novel genes expressed in Xenopus germ line: characteristic features of putative protein structures, their gene expression profiles and their possible roles in gametogenesis and embryogenesis.

We compared the secondary spermatogonia and the primary spermatocytes of Xenopus for the proteins in their microsomal fractions and identified a newly synthesized protein (94 kDa) and three other proteins (99, 85, and 72 kDa) which increased their amount after entering the meiotic phase. These four proteins were used as antigens to produce polyclonal antibody which was found to react with the four proteins as well as two other proteins (208 and 60 kDa). Immunoscreening of Xenopus testis cDNA library with this polyclonal antibody yielded two cDNA clones (Xmegs and Xtr) encoding novel proteins. Xmegs mRNA was specifically expressed in the spermatogenic cells from the mid-pachytene stage to completion of two meiotic divisions. The putative Xmegs protein contained 19 tandem repeats of 26 amino acid residues rich in proline as well as potential phosphorylation sites (i.e., serine and threonine residues). Around this repetitive area, we found five PEST sequences known as a proteolytic signal to target protein for degradation. The presence of PEST sequences was believed to allow protein levels to closely parallel mRNA abundance. These results suggested the possible role of this novel protein in the regulation of two meiotic divisions specific to the spermatogenesis in a phosphorylation- and/or dephosphorylation-dependent manner. On the other hand, Xtr mRNA was expressed in both spermatogenic and oogenic cells except for round spermatids and the later stage cells. This mRNA was also expressed in the early stage embryos and its amount was kept constant from the St. I oocyte to the gastrula stage and decreased thereafter. The putative Xtr protein contained four complete and one partial tudor-like domains that were discovered in Drosophila tudor protein which plays an important role in PGC differentiation and abdominal segmentation. The characteristic expression profile of Xtr and the protein structure similar to the Drosophila tudor protein suggested its possible role in the progression of meiosis and PGC differentiation.

Identification of tudor repeat associator with PCTAIRE 2 (Trap). A novel protein that interacts with the N-terminal domain of PCTAIRE 2 in rat brain.

PCTAIRE 2 is a Cdc2-related kinase that is predominantly expressed in the terminally differentiated neuron. To elucidate the function of PCTAIRE 2, proteins that associate with PCTAIRE 2 were screened by the yeast two-hybrid system. A positive clone was found to encode a novel protein that could bind to PCTAIRE 2 in vitro as well as in vivo, and was designated as Trap (tudor repeat associator with PCTAIRE 2). The overall structure of Trap shows no significant homology to any proteins, but contains five repeated domains (the tudor-like domain), conserved in Drosophila tudor protein. Trap associates with the N-terminal domain of PCTAIRE 2 through its C-terminal domain, which contains two tudor-like domains. PCTAIRE 1, but not PCTAIRE 3, can also associate with Trap. Trap is predominantly expressed in brain and testis, and gradually increases during brain development throughout life, consistent with the expression pattern of PCTAIRE 2. Immunoreactivities for PCTAIRE 2 and Trap were colocalized to the mitochondria in COS 7 cells. Immunohistochemical analyses showed that PCTAIRE 2 and Trap were distributed in the same cell layer of the cerebral cortex and cerebellum. These findings suggest that Trap is a physiological partner of PCTAIRE 2 in terminally differentiated neurons.