Galectin-3-U1 snRNP Complexes Initiate Splicing Activity in U1-Depleted Nuclear Extracts.

Fractionation of HeLa cell nuclear extracts by glycerol gradient centrifugation separates endogenous uracil-rich small nuclear ribonucleoprotein complexes (U snRNP) into numerous particles sedimenting from 7S to greater than 60S. Complexes sedimenting at 10S contain a single U snRNP (U1 snRNP) and galectin-3. Addition of antibodies specific for galectin-3 to fractions containing these 10S complexes coprecipitates U1 snRNP, indicating that a fraction of the U1 snRNP is associated with this galectin. Galectin-3 has been shown by depletion-reconstitution studies to be an integral splicing component involved both in spliceosome assembly and splicing activity. The first step in initiation of spliceosome assembly is binding of U1 snRNP to the 5' splice site of the premessenger RNA substrate. The finding that U1 snRNP and galectin-3 are associated in splicing extracts hints that this complex affords a potential entry point for galectin-3 into the splicing pathway. Addition of U1 snRNP-galectin-3 complexes immunoselected from the 10S region of glycerol gradients to a U1-depleted nuclear extract initiates splicing activity with the formation of splicing intermediates and mature mRNA. This chapter describes the materials and methods for these experiments that document galectin-3-U1 snRNP complexes initiate the splicing reaction in a U1-depleted nuclear extract.

Three-color intranuclear staining for measuring mitosis and apoptosis in cells transfected with a GFP-tagged histone.

Green fluorescent protein (GFP) has become a powerful tool for monitoring the expression of transfected genes by flow cytometry including GFP-tagged histones for tracking chromatin and elucidating histone function. We describe here a method for simultaneous detection of three nucleus-localized signals: a GFP-tagged histone, DNA content and detection of phosphorylated histone H3, which labels mitotic cells. We also demonstrate another application of this method for simultaneous detection of a GFP-tagged histone, DNA content, and cleaved caspase-3.

Phytochrome nuclear body: an emerging model to study interphase nuclear dynamics and signaling.

In higher plants, one of the most striking effects of light at the cellular level is the formation of phytochrome nuclear bodies (PNBs). In Arabidopsis, two types of PNBs have been described: a transient type of PNBs (tPNBs), containing both phytochrome A and phytochrome B, observed during the dark-to-light transition and a relatively photo-stable type of phytochrome B-containing PNBs (sPNBs) under continuous light. Despite the separation of the cell-biological observations of PNBs from the traditional model of light signaling elucidated by genetic and biochemical approaches, a growing body of evidence indicates that PNBs are intimately involved in phytochrome signaling. Both positive and negative light signaling components have been colocalized to PNBs, which provides direct evidence bridging PNBs and phytochrome signaling. In particular, the sPNB serves as an excellent tractable marker for early phytochrome signaling events, and thus provides a remarkable genetic system to investigate the mechanistic connection between interphase subnuclear dynamics and cell signaling.

Arabidopsis CBF5 interacts with the H/ACA snoRNP assembly factor NAF1.

The conserved protein CBF5, initially regarded as a centromere binding protein in yeast and higher plants, was later found within nucleoli and in Cajal bodies of yeast and metazoa. There, it is assumed to be involved in posttranscriptional pseudouridinylation of various RNA species that might be important for RNA processing. We found EYFP-labeled CBF5 of A. thaliana to be located within nucleoli and Cajal bodies, but neither at centromeres nor somewhere else on chromosomes. Arabidopsis mutants carrying a homozygous T-DNA insertion at the CBF5 locus were lethal. Yeast two-hybrid and mRNA expression analyses demonstrated that AtCBF5 is co-expressed and interacts with a previously uncharacterized protein containing a conserved NAF1 domain, presumably involved in H/ACA box snoRNP biogenesis. The homologous yeast protein has been shown to contribute to RNA pseudouridinylation. Thus, AtCBF5 might have an essential function in RNA processing rather than being a kinetochore protein.

Nucleoplasmic LAP2alpha-lamin A complexes are required to maintain a proliferative state in human fibroblasts.

In human diploid fibroblasts (HDFs), expression of lamina-associated polypeptide 2 alpha (LAP2alpha) upon entry and exit from G(0) is tightly correlated with phosphorylation and subnuclear localization of retinoblastoma protein (Rb). Phosphoisoforms of Rb and LAP2alpha are down-regulated in G(0). Although RbS780 phosphoform and LAP2alpha are up-regulated upon reentry into G(1) and colocalize in the nucleoplasm, RbS795 migrates between nucleoplasmic and speckle compartments. In HDFs, which are null for lamins A/C, LAP2alpha is mislocalized within nuclear aggregates, and this is correlated with cell cycle arrest and accumulation of Rb within speckles. Nuclear retention of nucleoplasmic Rb during G(1) phase but not of speckle-associated Rb depends on lamin A/C. siRNA knock down of LAP2alpha or lamin A/C in HDFs leads to accumulation of Rb in speckles and G(1) arrest, probably because of activation of a cell cycle checkpoint. Our results suggest that LAP2alpha and lamin A/C are involved in controlling Rb localization and phosphorylation, and a lack or mislocalization of either protein leads to cell cycle arrest in HDFs.

Intracellular trafficking of LET-756, a fibroblast growth factor of C. elegans, is controlled by a balance of export and nuclear signals.

The superfamily of fibroblast growth factors (FGF), which counts 22 members in humans, exerts many functions during animal development and adult life. LET-756 is one of the two FGFs of the nematode C. elegans. Re-introduction of LET-756 in a null mutant strain restores viability, allowing the study of structural requirements for LET-756 trafficking and function. LET-756 protein has several regions and motifs, including a non-classical internal motif required for secretion. We show here that a main difference in the wild-type LET-756 molecule and a truncated molecule that mimics a partial loss-of-function mutant lies on subnuclear expression. Using Cos-1 cells and rescue activity we show that: (i) nuclear localization is due to various redundant NLS, one of them acting as a nucleolar localization signal; (ii) nuclear LET-756 is addressed to the speckles by a stretch of glutamine residues; (iii) nuclear LET-756 is trafficking between speckles and nucleoli; (iv) in the nucleolus, LET-756 is associated with proteins of the rRNA splicing compartment; (v) changing LET-756 secretion signal prevents its nuclear localization. We propose that LET-756 exerts its functions through a balance between secreted and nuclear forms due to two opposite addressing signals, (i) synergy of several NLS and (ii) attenuated secretion signal.

Predicting protein subnuclear location with optimized evidence-theoretic K-nearest classifier and pseudo amino acid composition.

The nucleus is the brain of eukaryotic cells that guides the life processes of the cell by issuing key instructions. For in-depth understanding of the biochemical process of the nucleus, the knowledge of localization of nuclear proteins is very important. With the avalanche of protein sequences generated in the post-genomic era, it is highly desired to develop an automated method for fast annotating the subnuclear locations for numerous newly found nuclear protein sequences so as to be able to timely utilize them for basic research and drug discovery. In view of this, a novel approach is developed for predicting the protein subnuclear location. It is featured by introducing a powerful classifier, the optimized evidence-theoretic K-nearest classifier, and using the pseudo amino acid composition [K.C. Chou, PROTEINS: Structure, Function, and Genetics, 43 (2001) 246], which can incorporate a considerable amount of sequence-order effects, to represent protein samples. As a demonstration, identifications were performed for 370 nuclear proteins among the following 9 subnuclear locations: (1) Cajal body, (2) chromatin, (3) heterochromatin, (4) nuclear diffuse, (5) nuclear pore, (6) nuclear speckle, (7) nucleolus, (8) PcG body, and (9) PML body. The overall success rates thus obtained by both the re-substitution test and jackknife cross-validation test are significantly higher than those by existing classifiers on the same working dataset. It is anticipated that the powerful approach may also become a useful high throughput vehicle to bridge the huge gap occurring in the post-genomic era between the number of gene sequences in databases and the number of gene products that have been functionally characterized. The OET-KNN classifier will be available at www.pami.sjtu.edu.cn/people/hbshen.

Corepressor subnuclear organization is regulated by estrogen receptor via a mechanism that requires the DNA-binding domain.

The restriction of transcription factors to certain domains within the cell nucleus must serve an important regulatory function. The silencing mediator of retinoic acid and thyroid hormone (SMRT) and other members of the corepressor complex are enriched in spherical intranuclear foci, and repress estrogen receptor alpha (ERalpha)-dependent transcriptional activity. When fluorescent protein (FP)-labeled SMRT and ERalpha were co-expressed, the proteins co-localized. The subnuclear organization and positioning of the complexes, however, depended on the ligand state of the receptor. Automated image analysis was used to quantify the ERalpha-dependent change in SMRT organization in randomly selected living cell populations. The results demonstrate that the subnuclear positioning of SMRT is influenced by the ligand-bound ERalpha, and this activity is dependent on the ratio of the co-expressed ERalpha and SMRT. A deletion mutant of ERalpha showed that the receptor DNA-binding domain was necessary for the ligand-dependent positioning of SMRT. These results define important organizational mechanisms that underlie nuclear receptor regulation of gene expression.

A role for macromolecular crowding effects in the assembly and function of compartments in the nucleus.

The mechanisms which cause macromolecules to form discrete compartments within the nucleus are not understood. Here, two ubiquitous compartments, nucleoli, and PML bodies, are shown to disassemble when K562 cell nuclei expand in medium of low monovalent cation concentration; their major proteins dispersed as seen by immunofluorescence and immunoelectron microscopy, and nucleolar transcript elongation fell by approximately 85%. These compartments reassembled and nucleolar transcription recovered in the same medium after adding inert, penetrating macromolecules (8 kDa polyethylene glycol (PEG), or 10.5 kDa dextran) to 12% w/v, showing that disassembly was not caused by the low cation concentration. These responses satisfy the criteria for crowding or volume exclusion effects which occur in concentrated mixtures of macromolecules; upon expansion the macromolecular concentration within the nucleus falls, and can be restored by PEG or dextran. These observations, together with evidence of a high concentration of macromolecules in the nucleus (in the range of 100mg/ml) which must cause strong crowding forces, suggest strongly that these forces play an essential role in driving the formation, and maintaining the function of nuclear compartments. This view is consistent with their dynamic and mobile nature and can provide interpretations of several unexplained observations in nuclear biology.

Computer-assisted image analysis protocol that quantitatively measures subnuclear protein organization in cell populations.

Many nuclear proteins, including the nuclear receptor co-repressor (NCoR) protein are localized to specific regions of the cell nucleus, and this subnuclear positioning is preserved when NCoR is expressed in cells as a fusion to a fluorescent protein (FP). To determine how specific factors may influence the subnuclear organization of NCoR requires an unbiased approach to the selection of cells for image analysis. Here, we use the co-expression of the monomeric red FP (mRFP) to select cells that also express NCoR labeled with yellow FP (YFP). The transfected cells are selected for imaging based on the diffuse cellular mRFP signal without prior knowledge of the subnuclear organization of the co-expressed YFP-NCoR. The images acquired of the expressed FPs are then analyzed using an automated image analysis protocol that identifies regions of interest (ROIs) using a set of empirically determined rules. The relative expression levels of both fluorescent proteins are estimated, and YFP-NCoR subnuclear organization is quantified based on the mean focal body size and relative intensity. The selected ROIs are tagged with an identifier and annotated with the acquired data. This integrated image analysis protocol is an unbiased method for the precise and consistent measurement of thousands of ROIs from hundreds of individual cells in the population.

New type of snRNP containing nuclear bodies in plant cells.

In larch (Larix decidua Mill.) microspores a new type of nuclear bodies has been found which are an element of the spatial organization of the splicing system in plant cell. These are bizonal bodies, ultrastructurally differentiated into a coiled part and a dense part. Using immunocytochemistry and in situ hybridization at the EM level, the coiled part of the bizonal body was found to contain snRNA including U2 snRNA, Sm proteins and nucleolar proteins of the agyrophilic type and fibrillarin. The dense part contains Sm proteins but lacks snRNA. Such a separation of macromolecules related to splicing occurring within the bizonal bodies microspore is striking by the similarity of these bodies to amphibian oocyte snurposomes. The occurrence in plant cells, beside widely known coiled bodies (CBs), also of other nuclear bodies related to splicing proves that in plants similarly as for animals the differentiation among domains containing elements of the splicing system occurs.