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The branched architecture of neuronal dendrites is a key factor in how neurons form ordered networks and discoveries continue to be made identifying proteins and protein–protein interactions that direct or execute the branching and extension of dendrites. Our prior work showed that the molecular scaffold Pdlim5 and delta-catenin, in conjunction, are two proteins that help regulate the branching and elongation of dendrites in cultured hippocampal neurons and do so through a phosphorylation-dependent mechanism triggered by upstream glutamate signaling. In this report we have focused on Pdlim5's multiple scaffolding domains and how each contributes to dendrite branching. The three identified regions within Pdlim5 are the PDZ, DUF, and a trio of LIM domains; however, unresolved is the intra-molecular conformation of Pdlim5 as well as which domains are essential to regulate dendritic branching. We address Pdlim5's structure and function by examining the role of each of the domains individually and using deletion mutants in the context of the full-length protein. Results using primary hippocampal neurons reveal that the Pdlim5 DUF domain plays a dominant role in increasing dendritic branching. Neither the PDZ domain nor the LIM domains alone support increased branching. The central role of the DUF domain was confirmed using deletion mutants in the context of full-length Pdlim5. Guided by molecular modeling, additional domain mapping studies showed that the C-terminal LIM domain forms a stable interaction with the N-terminal PDZ domain, and we identified key amino acid residues at the interface of each domain that are needed for this interaction. We posit that the central DUF domain of Pdlim5 may be subject to modulation in the context of the full-length protein by the intra-molecular interaction between the N-terminal PDZ and C-terminal LIM domains. Overall, our studies reveal a novel mechanism for the regulation of Pdlim5's function in the regulation of neuronal branching and highlight the critical role of the DUF domain in mediating these effects. [ABSTRACT FROM AUTHOR]

Here we describe a spontaneous mutation in the Zdhhc13 (zinc finger, DHHC domain containing 13) gene (also called Hip14l), one of 24 genes encoding palmitoyl acyltransferase (PAT) enzymes in the mouse. This mutation (Zdhhc13luc) was identified as a nonsense base substitution, which results in a premature stop codon that generates a truncated form of the ZDHHC13 protein, representing a potential loss-of-function allele. Homozygous Zdhhc13luc/Zdhhc13luc mice developed generalized hypotrichosis, associated with abnormal hair cycle, epidermal and sebaceous gland hyperplasia, hyperkeratosis, and increased epidermal thickness. Increased keratinocyte proliferation and accelerated transit from basal to more differentiated layers were observed in mutant compared with wild-type (WT) epidermis in untreated skin and after short-term 12-O-tetradecanoyl-phorbol-13-acetate treatment and acute UVB exposure. Interestingly, this epidermal phenotype was associated with constitutive activation of NF-κB (RelA) and increased neutrophil recruitment and elastase activity. Furthermore, tumor multiplicity and malignant progression of papillomas after chemical skin carcinogenesis were significantly higher in mutant mice than WT littermates. To our knowledge, this is the first report of a protective role for PAT in skin carcinogenesis.

Human protein arginine methyltransferase (PRMT) 9 symmetrically dimethylates arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. Although amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylarginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides.

Tudor domain-containing protein 3 (TDRD3) is a major methylarginine effector molecule that reads methyl-histone marks and facilitates gene transcription. However, the underlying mechanism by which TDRD3 functions as a transcriptional coactivator is unknown. We identified topoisomerase IIIB (TOP3B) as a component of the TDRD3 complex. TDRD3 serves as a molecular bridge between TOP3B and arginine-methylated histones. The TDRD3-TOP3B complex is recruited to the c-MYC gene promoter primarily by the H4R3me2a mark, and the complex promotes c-MYC gene expression. TOP3B relaxes negative supercoiled DNA and reduces transcription-generated R loops in vitro. TDRD3 knockdown in cells increases R loop formation at the c-MYC locus, and Tdrd3 null mice exhibit elevated R loop formation at this locus in B cells. Tdrd3 null mice show significantly increased c-Myc/Igh translocation, a process driven by R loop structures. By reducing negative supercoiling and resolving R loops, TOP3B promotes transcription, protects against DNA damage, and reduces the frequency of chromosomal translocations.

The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7.