INCENP centromere and spindle targeting: identification of essential conserved motifs and involvement of heterochromatin protein HP1.

The inner centromere protein (INCENP) has a modular organization, with domains required for chromosomal and cytoskeletal functions concentrated near the amino and carboxyl termini, respectively. In this study we have identified an autonomous centromere- and midbody-targeting module in the amino-terminal 68 amino acids of INCENP. Within this module, we have identified two evolutionarily conserved amino acid sequence motifs: a 13-amino acid motif that is required for targeting to centromeres and transfer to the spindle, and an 11-amino acid motif that is required for transfer to the spindle by molecules that have targeted previously to the centromere. To begin to understand the mechanisms of INCENP function in mitosis, we have performed a yeast two-hybrid screen for interacting proteins. These and subsequent in vitro binding experiments identify a physical interaction between INCENP and heterochromatin protein HP1(Hsalpha). Surprisingly, this interaction does not appear to be involved in targeting INCENP to the centromeric heterochromatin, but may instead have a role in its transfer from the chromosomes to the anaphase spindle.

Involvement of U6 snRNA in 5' splice site selection.

Two models describing the interaction between U6 small nuclear RNA (snRNA) and the 5' splice site of introns have been proposed on the basis of cross-linking experiments. Here it is shown that a conserved sequence present in U6 snRNA forms base pairs with conserved nucleotides at the 5' splice junction and that this interaction is involved in 5' splice site choice. These results demonstrate a specific function for U6 snRNA in splicing and suggest that U6 snRNA has a proofreading role during splice site selection. A model is presented in which this new interaction, in concert with previously described interactions between U6 snRNA, U2 snRNA, and the pre-messenger RNA, would position the branch point near the 5' splice site for the catalysis of the first splicing step.

DNA topoisomerase IIalpha interacts with CAD nuclease and is involved in chromatin condensation during apoptotic execution.

Apoptotic execution is characterized by dramatic changes in nuclear structure accompanied by cleavage of nuclear proteins by caspases (reviewed in [1]). Cell-free extracts have proved useful for the identification and functional characterization of activities involved in apoptotic execution [2-4] and for the identification of proteins cleaved by caspases [5]. More recent studies have suggested that nuclear disassembly is driven largely by factors activated downstream of caspases [6]. One such factor, the caspase-activated DNase, CAD/CPAN/DFF40 [4,7,8] (CAD) can induce apoptotic chromatin condensation in isolated HeLa cell nuclei in the absence of other cytosolic factors [6,8]. As chromatin condensation occurs even when CAD activity is inhibited, however, CAD cannot be the sole morphogenetic factor triggered by caspases [6]. Here we show that DNA topoisomerase IIalpha (Topo IIalpha), which is essential for both condensation and segregation of daughter chromosomes in mitosis [9], also functions during apoptotic execution. Simultaneous inhibition of Topo IIalpha and caspases completely abolishes apoptotic chromatin condensation. In addition, we show that CAD binds to Topo IIalpha, and that their association enhances the decatenation activity of Topo IIalpha in vitro.

INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow.

Cytoskeletal rearrangements during mitosis must be co-ordinated with chromosome movements. The 'chromosomal passenger' proteins [1], which include the inner centromere protein (INCENP [2]), the Aurora-related serine-threonine protein kinase AIRK2 [3,4] and the unidentified human autoantigen TD-60 [5], have been suggested to integrate mitotic events. These proteins are chromosomal until metaphase but subsequently transfer to the midzone microtubule array and the equatorial cortex during anaphase. Disruption of INCENP function affects both chromosome segregation and completion of cytokinesis [6,7], whereas interference with AIRK2 function primarily affects cytokinesis [3,8]. Here, we report that INCENP is stockpiled in Xenopus eggs in a complex with Xenopus AIRK2 (XAIRK2), and that INCENP and AIRK2 kinase bind one another in vitro. This association was found to be evolutionarily conserved. Sli15p, the binding partner of yeast Aurora kinase Ipl1p, can be recognized as an INCENP family member because of the presence of a conserved carboxy-terminal sequence region, which we term the IN box. This interaction between INCENP and Aurora kinase was found to be biologically relevant. INCENP and AIRK2 colocalized exactly in human cells, and INCENP was required to target AIRK2 correctly to centromeres and the central spindle.

3' splice site recognition in S. cerevisiae does not require base pairing with U1 snRNA.

The conserved nucleotides 9 and 10 of U1 small nuclear RNA (snRNA) have been proposed to base pair with either 5' exon or 3' splice site sequences. In S. pombe, U1 snRNA pairing with the conserved 3' splice site is required for the first step of splicing and viability. In contrast, we show that S. cerevisiae U1 mutants at positions 9 and 10 are fully functional. Splicing of several genes is normal in these strains, ruling out an essential base pairing between U1 snRNA and 3' splice sites. U1 snRNA positions 9 and 10 are shown to be involved in 5' splice site selection through their interaction with exon sequences. Our results demonstrate that some snRNA-pre-mRNA interactions are not evolutionarily conserved and that 3' splice site recognition occurs by different mechanisms in various organisms.

An efficient PCR mutagenesis strategy without gel purification [correction of "purificiation"] step that is amenable to automation.

We describe here an improved megaprimer PCR mutagenesis strategy. The cumbersome gel purification step that is usually used can be omitted by appropriately cleaving the first and second DNA templates with restriction enzymes and enzymatically removing remaining primers from the first PCR reaction. We show that this improved procedure is reproducible and highly efficient. Furthermore this method is suitable for automation because all the steps are now carried out in reaction tubes.

A family of Ran binding proteins that includes nucleoporins.

Ran, a small nuclear GTP binding protein, is essential for the translocation of nuclear proteins through the nuclear pore complex. We show that several proteins, including the Saccharomyces cerevisiae Nup2p and Caenorhabditis elegans F59A2.1 nucleoporins, contain domains similar to the previously characterized murine Ran binding protein (RBP, termed RBP1). To test the significance of this similarity, we have used the corresponding domains of Nup2p and a putative S. cerevisiae RBP in Ran binding assays and the yeast two-hybrid system. Both proteins bind S. cerevisiae Ran, but only the putative S. cerevisiae RBP binds human Ran. Two-hybrid analysis revealed Ran-Ran interactions and that yeast and human Rans can interact. These data identify Nup2p as a target for Ran in the nuclear pore complex, suggesting a direct role for it in nuclear-cytoplasmic transport. We discuss the possibility that proteins harboring Ran binding domains link the Ran GTPase cycle to specific functions in the nucleus.

Drosophila SNF/D25 combines the functions of the two snRNP proteins U1A and U2B' that are encoded separately in human, potato, and yeast.

The plant and vertebrate snRP proteins U1A and U2B' are structurally closely related, but bind to different U snRNAs. Two additional related snRNP proteins, the yeast U2B' protein and Drosophila SNF/D25 protein, are analyzed here. We show that the previously described yeast open reading frame YIB9w encodes yeast U2B' as judged by the fact that the protein encoded by YIB9w bindsto stem-loop IV of yeast U2 snRNA in vitro and is part of the U2 snRNP in vivo. In contrast to the human U2B' protein, specific binding of yeast U2B' to RNA in vitro can occur in the absence of an accessory U2A' protein. The Drosophila SNF-D25 protein, unlike all other U1A/U2B' proteins studied to date, is shown to be a component of both U1 and U2 snRNPs. In vitro, SNF/D25 binds to U1 snRNA on itsown and to U2 snRNA in the presence of either the human U2A' protein or of Drosophila nuclear extract. Thus, its RNA-binding properties are the sum of those exhibited by human or potato U1A and U2B' proteins. Implications for the role of SNF/D25 in alternative splicing, and for the evolution of the U1A/U2B' protein family, are discussed.

Sm and Sm-like proteins assemble in two related complexes of deep evolutionary origin.

A group of seven Sm proteins forms a complex that binds to several RNAs in metazoans. All Sm proteins contain a sequence signature, the Sm domain, also found in two yeast Sm-like proteins associated with the U6 snRNA. We have performed database searches revealing the presence of 16 proteins carrying an Sm domain in the yeast genome. Analysis of this protein family confirmed that seven of its members, encoded by essential genes, are homologues of metazoan Sm proteins. Immunoprecipitation revealed that an evolutionarily related subgroup of seven Sm-like proteins is directly associated with the nuclear U6 and pre-RNase P RNAs. The corresponding genes are essential or required for normal vegetative growth. These proteins appear functionally important to stabilize U6 snRNA. The two last yeast Sm-like proteins were not found associated with RNA, and neither was essential for vegetative growth. To investigate whether U6-associated Sm-like protein function is widespread, we cloned several cDNAs encoding homologous human proteins. Two representative human proteins were shown to associate with U6 snRNA-containing complexes. We also identified archaeal proteins related to Sm and Sm-like proteins. Our results demonstrate that Sm and Sm-like proteins assemble in at least two functionally conserved complexes of deep evolutionary origin.

New constructs and strategies for efficient PCR-based gene manipulations in yeast.

Gene disruption and tagging can be achieved by homologous recombination in the yeast genome. Several PCR-based methods have been described towards this end. However these strategies are often limited in their applications and/or their efficiencies and may be technically demanding. Here we describe two plasmids for C-terminal tagging of proteins with the IgG binding domain of the Staphylococcus aureus protein A. We also present simple and reliable strategies based on PCR to promote efficient integration of exogenous DNA into the yeast genome. These simple methods are not limited to specific strains or markers and can be used for any application requiring homologous recombination such as gene disruption and epitope tagging. These strategies can be used for consecutive introduction of various constructs into a single yeast strain.

INCENP binds directly to tubulin and requires dynamic microtubules to target to the cleavage furrow.

Inner centromere protein (INCENP) is a chromosomal passenger protein with an essential role in mitosis. At the metaphase/anaphase transition, some INCENP transfers from the centromeres to the central spindle; the remainder then transfers to the equatorial cortex prior to cleavage furrow formation. The molecular associations dictating INCENP behavior during mitosis are currently unknown. Here we show that targeting INCENP to the cleavage plane requires dynamic microtubules, but not F-actin. When microtubules are eliminated, INCENP is dispersed across the entire cell cortex. Yeast two-hybrid and in vitro binding data demonstrate that INCENP binds directly to beta-tubulin via a conserved domain encompassing residues 48-85. Furthermore, INCENP binds to microtubules polymerized from purified tubulin in vitro and appears to bundle microtubules when expressed in the interphase cytoplasm. These data indicate that INCENP is a microtubule-binding protein that targets to the equatorial cortex through interactions requiring microtubules.