2013;24:100C112. of specific PRMTs and their target proteins in flagella and demonstrate that PRMTs are cargo for translocation within flagella by the process of IFT. INTRODUCTION Cilia and flagella (here used as interchangeable terms) are well-conserved organelles that project from the cell body and are found on diverse eukaryotic cell types from unicellular protists to human cells. Flagella Yunaconitine comprise 600 different polypeptides (Pazour 2005 ), and defects in motile cilia can Yunaconitine be the cause of a heterogeneous set of human phenotypes collectively called primary ciliary dyskinesia (Afzelius, 2004 ; Lee, 2011 ). Sliding of flagellar doublet microtubules via the dynein arms is indispensable for motility (Summers and Gibbons, 1971 ), which is usually controlled by the radial spokes and central pair apparatus (Smith and Yang, 2004 ). Collectively the outer doublets, central pair apparatus, radial spokes, and dynein arms comprise the axoneme, a protein complex that is held together by noncovalent interactions. In addition to the flagella of sperm and the motile cilia of the airway, fallopian Yunaconitine tubes, efferent ducts of the rete testis, and the ependyma of the brain, a single nonmotile cilium lacking the central apparatus, spokes, and arms, called the primary cilium, is also present on almost all mammalian cell types. Defects in primary cilia are associated with many human disorders, collectively referred to as ciliopathies. Some are relatively minor in effect (e.g., polydactyly), whereas others are much more severe (e.g., polycystic kidney disease, Joubert syndrome; Tobin and Beales, 2009) . Assembly, disassembly, and turnover of the axoneme are dependent on a unique motility process called intraflagellar transport (IFT; Kozminski flagella Mouse monoclonal to LPL and showed that protein methylation on arginine residues is usually up-regulated during flagellar resorption (Schneider genome encodes at least seven PRMTs, four of which we study in detail here (type I PRMT 1, 3, and 10, and PRMT 5, a type II enzyme). These four enzymes are present in a punctate pattern along the length of the flagella, similar to, but not entirely coincident with, the distribution of IFT particles; the PRMTs are also enriched at the flagellar tip. In addition to tip localization, PRMT 1 and 3 also exhibit very strong localization to the flagellar base in the region of the transition zone, and this basal localization changes during the processes of flagellar resorption and regeneration. Proteins with aDMA modifications Yunaconitine were also detected at the base and the tip of flagella, as well as in a punctate pattern of distribution comparable to that of the PRMTs. Furthermore, the data show that PRMTs are transported by IFT, during flagellar resorption and that the PRMTs can hop onto and off IFT trains at local stations along the length of the flagellum. Our results strongly indicate that PRMTs and aDMA modifications of axonemal proteins in the flagella play key roles Yunaconitine in both the assembly and disassembly processes, and it is possible that the process of IFT requires, or is regulated at least in part by, protein methylation. RESULTS The genome encodes at least seven PRMTs The mammalian genome encodes 10 PRMTs (Bedford and Clarke 2009 ), which either have been shown to produce dimethyl arginine (PRMT 1, 3C6, and 8) or are suspected of having this activity based on sequence analysis (PRMT 2, 7, and 9C11). PRMT 5 is usually a type II enzyme that generates sDMA; the others with exhibited methylation activity are type I enzymes that produce aDMA. To identify the potential PRMT genes in the genome, we performed a TBLASTN search of the database (JGI ver5.5), using as query sequences human PRMT 1C3, CARM1 (PRMT4), and PRMT 6C9. The identified sequences were then aligned with PRMT sequences from human, sea urchin, yeast, PRMT 1, 3, and 5 were clearly grouped with orthologous.