The centromere/kinetochore complex is indispensable for accurate segregation of chromosomes during cell divisions when it serves as the attachment site for spindle microtubules. Centromere identity in metazoans is believed to be governed by epigenetic mechanisms, because the highly repetitive centromeric DNA is neither sufficient nor required for specifying the assembly site of the kinetochore. A candidate for an epigenetic mark is the centromere-specific histone H3 variant CENP-A that replaces H3 in alternating blocks of chromatin exclusively in active centromeres. CENP-A acts as an initiator of kinetochore assembly, but the detailed dynamics of the deposition of metazoan CENP-A and of other constitutive kinetochore components are largely unknown. Here we show by quantitative fluorescence measurements in living early embryos that functional fluorescent fusion proteins of the Drosophila CENP-A and CENP-C homologs are rapidly incorporated into centromeres during anaphase. This incorporation is independent of ongoing DNA synthesis and pulling forces generated by the mitotic spindle, but strictly coupled to mitotic progression. Thus, our findings uncover a strikingly dynamic behavior of centromere components in anaphase.

Deletion of uridylates from the 3'-most editing site of synthetic ATPase 6 pre-mRNA can be visualized directly by coincubation of a radiolabeled substrate RNA and a synthetic gRNA in 20S fractions of T.brucie mitochondrial lysates. Substrate RNA cleavage is gRNA directed and occurs 3' to the uridylates to be deleted. U residues appear to be sequentially removed from the 3' end of the 5' cleavage product prior to religation of the two pre-mRNA halves. gRNA/mRNA chimeric molecules are also produced. Time course experiments indicate that chimeras appear after cleavage intermediates and edited product. Furthermore, a mutant gRNA promotes formation of edited product but not detectable chimeras. Our results suggest a model for kinetoplastid RNA editing in which chimeric molecules are nonproductive end products of editing and not intermediates that serve as a repository for deleted U's.

SummaryCohesin is a highly conserved multisubunit complex that holds sister chromatids together in mitotic cells. At the metaphase to anaphase transition, proteolytic cleavage of the α kleisin subunit (Rad21) by separase causes cohesin's dissociation from chromosomes and triggers sister-chromatid disjunction. To investigate cohesin's function in postmitotic cells, where it is widely expressed, we have created fruit flies whose Rad21 can be cleaved by TEV protease. Cleavage causes precocious separation of sister chromatids and massive chromosome missegregation in proliferating cells, but not disaggregation of polytene chromosomes in salivary glands. Crucially, cleavage in postmitotic neurons is lethal. In mushroom-body neurons, it causes defects in axon pruning, whereas in cholinergic neurons it causes highly abnormal larval locomotion. These data demonstrate essential roles for cohesin in nondividing cells and also introduce a powerful tool by which to investigate protein function in metazoa.

In the mitochondria of trypanosomatid protozoa the precursors of messenger RNAs (pre-mRNAs) have their coding information remodeled by the site-specific insertion and deletion of uridylate (U) residues. Small trans-acting guide RNAs (gRNAs) supply the genetic information for this RNA editing. An in vitro system was developed to study the mechanism of U insertion into pre-mRNA. U-insertion editing occurs through a series of enzymatic steps that begin with gRNA-directed pre-mRNA cleavage. Inserted U's are derived from free uridine triphosphate and are added to the 3' terminus of a 5' pre-mRNA cleavage product. gRNA specifies edited RNA sequence at the subsequent ligation step by base pairing-mediated juxtaposition of the 3' cleavage product and the processed 5' cleavage product. gRNA/pre-mRNA chimeras, purported intermediates, seem to be abortive end products of the same reaction.

Chromosome segregation during meiosis and mitosis depends on the assembly of functional kinetochores within centromeric regions. Centromeric DNA and kinetochore proteins show surprisingly little sequence conservation despite their fundamental biological role. However, our identification in Drosophila melanogaster of the most diverged orthologs identified so far, which encode components of a kinetochore protein network including the Ndc80 and Mis complexes, further emphasizes the notion of a shared eukaryotic kinetochore design. To determine its spatial organization, we have analyzed by quantitative light microscopy hundreds of native chromosomes from transgenic Drosophila strains coexpressing combinations of red and green fluorescent fusion proteins, fully capable of providing the essential wild-type functions. Thereby, Cenp-A/Cid, Cenp-C, Mis12 and the Ndc80 complex were mapped along the inter sister kinetochore axis with a resolution below 10 nm. The C terminus of Cenp-C was found to be near but well separated from the innermost component Cenp-A/Cid. The N terminus of Cenp-C is further out, clustered with Mis12 and the Spc25 end of the rod-like Ndc80 complex, which is known to bind to microtubules at its other more distal Ndc80/Nuf2 end.