Two different condensin complexes make distinct contributions to metaphase chromosome architecture in vertebrate cells. We show here that the spatial and temporal distributions of condensins I and II are differentially regulated during the cell cycle in HeLa cells. Condensin II is predominantly nuclear during interphase and contributes to early stages of chromosome assembly in prophase. In contrast, condensin I is sequestered in the cytoplasm from interphase through prophase and gains access to chromosomes only after the nuclear envelope breaks down in prometaphase. The two complexes alternate along the axis of metaphase chromatids, but they are arranged into a unique geometry at the centromere/kinetochore region, with condensin II enriched near the inner kinetochore plate. This region-specific distribution of condensins I and II is severely disrupted upon depletion of Aurora B, although their association with the chromosome arm is not. Depletion of condensin subunits causes defects in kinetochore structure and function, leading to aberrant chromosome alignment and segregation. Our results suggest that the two condensin complexes act sequentially to initiate the assembly of mitotic chromosomes and that their specialized distribution at the centromere/kinetochore region may play a crucial role in placing sister kinetochores into the back-to-back orientation. INTRODUCTIONThe faithful segregation of duplicated genetic information into two daughter cells is central to cell proliferation. In eukaryotic cells, amorphous interphase chromatin is converted into individual chromosomes composed of a pair of cylindrical structures (sister chromatids) by metaphase. This process is followed by synchronous segregation of sister chromatids that initiates at the onset of anaphase. During the past decade, a variety of approaches have been combined to demonstrate that a large protein complex called condensin is one of the key regulators of chromosome behavior during mitosis (reviewed by Nasmyth, 2002;Swedlow and Hirano, 2003).The condensin complex is composed of five subunits that are widely conserved among eukaryotic organisms from yeast to humans Sutani et al., 1999;Freeman et al., 2000;Schmiesing et al., 2000;Kimura et al., 2001). The two core subunits of condensin (CAP-E/SMC2 and CAP-C/SMC4) belong to a large family of chromosomal ATPases known as the structural maintenance of chromosomes (SMC) family. The SMC proteins participate in many aspects of higher order chromosome dynamics that include not only chromosome condensation but also sister chromatid cohesion and recombinational repair (reviewed by Hirano, 2002;Jessberger, 2002;Hagstrom and Meyer, 2003). The remaining subunits (CAP-D2, -G, and -H) are not related with SMCs, and share structural motifs with some components involved in cohesion (Neuwald and Hirano, 2000;Schleiffer et al., 2003). The holo-complex of condensin introduces positive superhelical tension into DNA in an ATP hydrolysis-dependent manner in vitro (Kimura and Hirano, 1997;Bazett-Jones et al., 2002), and ...

MicroRNAs (miRNAs) are important for regulating gene expression in muticellular organisms. MiRNA processing is a two-step process. In animal cells, the first step is nuclear and the second step cytoplasmic, whereas in plant cells, both steps occur in the nucleus via the enzyme Dicer-like1 (DCL1) and other proteins including the zinc-finger-domain protein Serrate (SE) and a double-stranded RNA (dsRNA) binding-domain protein, Hyponastic Leaves1 (HYL1). Furthermore, plant miRNAs are methylated by Hua Enhancer (HEN1) at their 3' ends and loaded onto Argonaute1 (AGO1). However, little is known about the cellular basis of miRNA biogenesis. Using live-cell imaging, we show here that DCL1 and HYL1 colocalize in discrete nuclear bodies in addition to being present in a low-level diffuse nucleoplasmic distribution. These bodies, which we refer to as nuclear dicing bodies (D-bodies), differ from Cajal bodies. A mutated DCL1 with impaired function in miRNA processing fails to target to D-bodies, and an introduced primary (pri)-miRNA transcript is recruited to D-bodies. Furthermore, bimolecular fluorescence complementation (BiFC) shows that DCL1, HYL1, and SE interact in D-bodies. On the basis of these data, we propose that D-bodies are crucial for orchestrating pri-miRNA processing and/or storage/assembly of miRNA-processing complexes in the nuclei of plant cells.

Centromeres interact with the spindle apparatus to enable chromosome disjunction and typically contain thousands of tandemly arranged satellite repeats interspersed with retrotransposons. While their role has been obscure, centromeric repeats are epigenetically modified and centromere specification has a strong epigenetic component. In the yeast Schizosaccharomyces pombe, long heterochromatic repeats are transcribed and contribute to centromere function via RNA interference (RNAi). In the higher plant Arabidopsis thaliana, as in mammalian cells, centromeric satellite repeats are short (180 base pairs), are found in thousands of tandem copies, and are methylated. We have found transcripts from both strands of canonical, bulk Arabidopsis repeats. At least one subfamily of 180–base pair repeats is transcribed from only one strand and regulated by RNAi and histone modification. A second subfamily of repeats is also silenced, but silencing is lost on both strands in mutants in the CpG DNA methyltransferase MET1, the histone deacetylase HDA6/SIL1, or the chromatin remodeling ATPase DDM1. This regulation is due to transcription from Athila2 retrotransposons, which integrate in both orientations relative to the repeats, and differs between strains of Arabidopsis. Silencing lost in met1 or hda6 is reestablished in backcrosses to wild-type, but silencing lost in RNAi mutants and ddm1 is not. Twenty-four–nucleotide small interfering RNAs from centromeric repeats are retained in met1 and hda6, but not in ddm1, and may have a role in this epigenetic inheritance. Histone H3 lysine-9 dimethylation is associated with both classes of repeats. We propose roles for transcribed repeats in the epigenetic inheritance and evolution of centromeres.

The organization and dynamics of the genome have been shown to influence gene expression in many organisms. Data from mammalian tissue culture cells have provided conflicting conclusions with regard to the extent to which chromatin organization is inherited from mother to daughter nuclei. To gain insight into chromatin organization and dynamics, we developed transgenic Arabidopsis lines in which centromeres were tagged with a green fluorescent protein fusion of the centromere-specific histone H3. Using four-dimensional (4-D) live cell imaging, we show that Arabidopsis centromeres are constrained at the nuclear periphery during interphase and that the organization of endoreduplicated sister centromeres is cell type dependent with predominant clustering in root epidermal cells and dispersion in leaf epidermal cells. 4-D tracking of the entire set of centromeres through mitosis, in growing root meristematic cells, demonstrated that global centromere position is not precisely transmitted from the mother cell to daughter cells. These results provide important insight into our understanding of chromatin organization among different cells of a living organism.

Serine/arginine-rich (SR) proteins are important splicing factors which play significant roles in spliceosome assembly and splicing regulation. However, little is known regarding their biological functions in plants. Here, we analyzed the phenotypes of mutants upon depleting different subfamilies of Arabidopsis SR proteins. We found that loss of the functions of SC35 and SC35-like (SCL) proteins cause pleiotropic changes in plant morphology and development, including serrated leaves, late flowering, shorter roots and abnormal silique phyllotaxy. Using RNA-seq, we found that SC35 and SCL proteins play roles in the pre-mRNA splicing. Motif analysis revealed that SC35 and SCL proteins preferentially bind to a specific RNA sequence containing the AGAAGA motif. In addition, the transcriptions of a subset of genes are affected by the deletion of SC35 and SCL proteins which interact with NRPB4, a specific subunit of RNA polymerase II. The splicing of FLOWERING LOCUS C (FLC) intron1 and transcription of FLC were significantly regulated by SC35 and SCL proteins to control Arabidopsis flowering. Therefore, our findings provide mechanistic insight into the functions of plant SC35 and SCL proteins in the regulation of splicing and transcription in a direct or indirect manner to maintain the proper expression of genes and development.