N 6 -Methyladenosine is a ubiquitous modification identified in the mRNA of numerous eukaryotes, where it is present within both coding and noncoding regions. However, this base modification does not alter the coding capacity, and its biological significance remains unclear. We show that Arabidopsis thaliana mRNA contains N 6 -methyladenosine at levels similar to those previously reported for animal cells. We further show that inactivation of the Arabidopsis ortholog of the yeast and human mRNA adenosine methylase (MTA) results in failure of the developing embryo to progress past the globular stage. We also demonstrate that the arrested seeds are deficient in mRNAs containing N 6 -methyladenosine. Expression of MTA is strongly associated with dividing tissues, particularly reproductive organs, shoot meristems, and emerging lateral roots. Finally, we show that MTA interacts in vitro and in vivo with At FIP37, a homolog of the Drosophila protein FEMALE LETHAL2D and of human WILMS' TUMOUR1-ASSOCIATING PROTEIN. The results reported here provide direct evidence for an essential function for N 6 -methyladenosine in a multicellular eukaryote, and the interaction with At FIP37 suggests possible RNA processing events that might be regulated or altered by this base modification.

POT1 is a single-copy gene in yeast and humans that encodes a single-strand telomere binding protein required for chromosome end protection and telomere length regulation. In contrast, Arabidopsis harbors multiple, divergent POT-like genes that bear signature N-terminal OB-fold motifs, but otherwise share limited sequence similarity. Here, we report that plants null for AtPOT1 show no telomere deprotection phenotype, but rather exhibit progressive loss of telomeric DNA. Genetic analysis indicates that AtPOT1 acts in the same pathway as telomerase. In vitro levels of telomerase activity in pot1 mutants are significantly reduced and are more variable than wild-type. Consistent with this observation, AtPOT1 physically associates with active telomerase particles. Although low levels of AtPOT1 can be detected at telomeres in unsynchronized cells and in cells arrested in G2, AtPOT1 binding is significantly enhanced during S-phase, when telomerase is thought to act at telomeres. Our findings indicate that AtPOT1 is a novel accessory factor for telomerase required for positive telomere length regulation, and they underscore the coordinate and extraordinarily rapid evolution of telomere proteins and the telomerase enzyme.

The FKBP12 (FK506-binding protein 12 kD) immunophilin interacts with several protein partners in mammals and is a physiological regulator of the cell cycle. In Arabidopsis, only one specific partner of AtFKBP12, namely AtFIP37 (FKBP12 interacting protein 37 kD), has been identified but its function in plant development is not known. We present here the functional analysis of AtFIP37 in Arabidopsis. Knockout mutants of AtFIP37 show an embryo-lethal phenotype that is caused by a strong delay in endosperm development and embryo arrest. AtFIP37 promoter::b-glucuronidase reporter gene constructs show that the gene is expressed during embryogenesis and throughout plant development, in undifferentiating cells such as meristem or embryonic cells as well as highly differentiating cells such as trichomes. A translational fusion with the enhanced yellow fluorescent protein indicates that AtFIP37 is a nuclear protein localized in multiple subnuclear foci that show a speckled distribution pattern. Overexpression of AtFIP37 in transgenic lines induces the formation of large trichome cells with up to six branches. These large trichomes have a DNA content up to 256C, implying that these cells have undergone extra rounds of endoreduplication. Altogether, these data show that AtFIP37 is critical for life in Arabidopsis and implies a role for AtFIP37 in the regulation of the cell cycle as shown for FKBP12 and TOR (target of rapamycin) in mammals.Immunophilins are a family of enzymes with a peptidyl-prolyl cis-trans isomerase activity (PPiase) involved in the folding of target proteins (Kay, 1996;Schiene-Fischer and Yu, 2001;Hur and Bruice, 2002;Shaw, 2002). Their function has been primarily studied in human immune response as they are receptors for immunosuppressive drugs. Among immunophilins, FK506-binding proteins (FKBPs) are intracellular receptors for the two related drugs, FK506 and rapamycin. FKBPs are found in many organisms, including prokaryotes, animals, and plants (Harrar et al., 2001;Breiman and Camus, 2002).While FKBPs differ in size, FKBP12 (12 kD) represents the minimal peptide sequence harboring the two main properties of FKBPs, namely the PPiase activity and drug binding. FKBP12 is a ubiquitous and abundant protein localized in the cytosol of mammalian cells (Maki et al., 1990). In mammals, FKBP12 is essential since a knockout mouse dies during embryonic development (Shou et al., 1998), while in yeast (Saccharomyces cerevisiae) loss of FKBP12 function does not affect cell viability (Dolinski et al., 1997). In the absence of drugs, FKBP12 is associated with receptors such as the type II-TGFb (transforming growth factor b) receptor or calcium channels such as the ryanodine receptor or the inositol-(1,4,5)-triphosphate receptor (Ins(1,4,5)P 3 R). When bound to FK506, FKBP12 dissociates from the receptors resulting in a misregulation of receptor activities. For instance, the activity of the TGFb receptor is leaky in the absence of ligand after the release of FKBP12 by FK506 leading to the inactivation by dephosphoryla...