Essential roles for Caenorhabditis elegans lamin gene in nuclear organization, cell cycle progression, and spatial organization of nuclear pore complexes.

Caenorhabditis elegans has a single lamin gene, designated lmn-1 (previously termed CeLam-1). Antibodies raised against the lmn-1 product (Ce-lamin) detected a 64-kDa nuclear envelope protein. Ce-lamin was detected in the nuclear periphery of all cells except sperm and was found in the nuclear interior in embryonic cells and in a fraction of adult cells. Reductions in the amount of Ce-lamin protein produce embryonic lethality. Although the majority of affected embryos survive to produce several hundred nuclei, defects can be detected as early as the first nuclear divisions. Abnormalities include rapid changes in nuclear morphology during interphase, loss of chromosomes, unequal separation of chromosomes into daughter nuclei, abnormal condensation of chromatin, an increase in DNA content, and abnormal distribution of nuclear pore complexes (NPCs). Under conditions of incomplete RNA interference, a fraction of embryos escaped embryonic arrest and continue to develop through larval life. These animals exhibit additional phenotypes including sterility and defective segregation of chromosomes in germ cells. Our observations show that lmn-1 is an essential gene in C. elegans, and that the nuclear lamins are involved in chromatin organization, cell cycle progression, chromosome segregation, and correct spacing of NPCs.

C. elegans nuclear envelope proteins emerin, MAN1, lamin, and nucleoporins reveal unique timing of nuclear envelope breakdown during mitosis.

Emerin, MAN1, and LAP2 are integral membrane proteins of the vertebrate nuclear envelope. They share a 43-residue N-terminal motif termed the LEM domain. We found three putative LEM domain genes in Caenorhabditis elegans, designated emr-1, lem-2, and lem-3. We analyzed emr-l, which encodes Ce-emerin, and lem-2, which encodes Ce-MAN1. Ce-emerin and Ce-MAN1 migrate on SDS-PAGE as 17- and 52-kDa proteins, respectively. Based on their biochemical extraction properties and immunolocalization, both Ce-emerin and Ce-MAN1 are integral membrane proteins localized at the nuclear envelope. We used antibodies against Ce-MAN1, Ce-emerin, nucleoporins, and Ce-lamin to determine the timing of nuclear envelope breakdown during mitosis in C. elegans. The C. elegans nuclear envelope disassembles very late compared with vertebrates and Drosophila. The nuclear membranes remained intact everywhere except near spindle poles during metaphase and early anaphase, fully disassembling only during mid-late anaphase. Disassembly of pore complexes, and to a lesser extent the lamina, depended on embryo age: pore complexes were absent during metaphase in >30-cell embryos but existed until anaphase in 2- to 24-cell embryos. Intranuclear mRNA splicing factors disassembled after prophase. The timing of nuclear disassembly in C. elegans is novel and may reflect its evolutionary position between unicellular and more complex eukaryotes.

The spatial and temporal dynamics of Sax1 (CHox3) homeobox gene expression in the chick's spinal cord.

Sax1 (previously CHox3) is a chicken homeobox gene belonging to the same homeobox gene family as the Drosophila NK1 and the honeybee HHO genes. Sax1 transcripts are present from stage 2 H&H until at least 5 days of embryonic development. However, specific localization of Sax1 transcripts could not be detected by in situ hybridization prior to stage 8-, when Sax1 transcripts are specifically localized in the neural plate, posterior to the hindbrain. From stages 8- to 15 H&H, Sax1 continues to be expressed only in the spinal part of the neural plate. The anterior border of Sax1 expression was found to be always in the transverse plane separating the youngest somite from the yet unsegmented mesodermal plate and to regress with similar dynamics to that of the segregation of the somites from the mesodermal plate. The posterior border of Sax1 expression coincides with the posterior end of the neural plate. In order to study a possible regulation of Sax1 expression by its neighboring tissues, several embryonic manipulation experiments were performed. These manipulations included: removal of somites, mesodermal plate or notochord and transplantation of a young ectopic notochord in the vicinity of the neural plate or transplantation of neural plate sections into the extraembryonic area. The results of these experiments revealed that the induction of the neural plate by the mesoderm has already occurred in full primitive streak embryos, after which Sax1 is autonomously regulated within the spinal part of the neural plate.

Possible involvement of (2'5')oligoadenylate synthetase activity in pre-mRNA splicing.

The first step in splicing of pre-mRNA involves an intermediate lariat structure, in which a 2'5' phosphodiester bond between the 5' terminal guanosine residue of the intron and a specific adenosine residue near the 3' end of the intron is formed. A mammalian enzyme that generates 2'-5' phosphodiester bonds is (2'-5')oligoadenylate synthetase [(2'-5')OASE]. Although the expression of this enzyme is induced by interferon, low constitutive levels can be detected in untreated cells and tissues. The structural similarity between the lariat branch point and the 2'-5' phosphodiester bond generated by (2'-5')OASE prompted the experiments described here which suggest that this enzyme is involved in pre-mRNA splicing. (i) We show that a (2'-5')OASE activity is associated with 60S spliceosomes in an ATP- and RNA-dependent manner and that it can be indirectly immunoprecipitated by anti-Sm antibodies. (ii) Antibodies against (2'-5')OASE inhibit the lariat formation in the first step of splicing when added directly to a splicing reaction in vitro. (iii) HeLa cell nuclear extracts immunodepleted of (2'-5')OASE activity were also deficient in splicing activity.

Isolation and visualization of large compact ribonucleoprotein particles of specific nuclear RNAs.

We have previously shown that nuclear transcripts of the multifunctional enzyme, carbamoyl-phosphate synthetase, aspartate transcarbamylase, dihydroorotase RNA can be released from nuclei of Syrian hamster cells as compact ribonucleoprotein (RNP) particles that sediment at the 200S region in a sucrose gradient. The 200S nuclear RNP particles contain U1, U2, and U6 small nuclear RNPs, which are known to be required for splicing of pre-mRNA, as integral components of the particles. In this study we demonstrate that nuclear transcripts of dihydrofolate reductase in Syrian hamster cells and of beta-actin in both Syrian hamster and human cells are also released from the respective nuclei as 200S particles--despite the difference in length of these RNAs. Electron microscopy of the 200S particles revealed discrete compact composite structures with a cross section of approximately 50 nm. Finding that two more nuclear RNAs from two different cell types and two different species are released as 200S RNP particles suggests a general mode for packaging of heterogeneous nuclear RNA in large compact RNP particles the size of which is independent of the RNA length.

U1, U2, and U6 small nuclear ribonucleoproteins (snRNPs) are associated with large nuclear RNP particles containing transcripts of an amplified gene in vivo.

Nuclear ribonucleoprotein (RNP) complexes that contain intact transcripts of the amplified gene for CAD, the multifunctional protein that initiates UMP synthesis in Syrian hamster cells, have been released from nuclei of Syrian hamster cells as large particulate structures that sediment at the 200S region in a sucrose gradient. By the technique of RNA hybridization, we have shown that U1, U2, and U6 small nuclear RNAs (snRNAs) cosediment with the large RNP particles in the sucrose gradients. Autoimmune sera from systemic lupus erythematosus and mixed connective tissue disease patients, characterized as anti-(U1)RNP, have further been shown to immunoprecipitate CAD RNA along with U1 and U2 snRNAs from the fractionated nuclear 200S RNP particles. We conclude that U1, U2, and U6 snRNPs are integral constituents of the 200S RNP particles. The requirement of snRNPs for RNA processing that evidently occurs on RNP particles has been recently demonstrated. Our results thus suggest that the 200S RNPs are structurally and functionally close to the native particles on which RNA processing occurs.

Abundant nuclear ribonucleoprotein form of CAD RNA.

Transcripts of the CAD gene in Syrian hamster cells are as abundant in the nucleus as in the cytoplasm. This was shown by in situ hybridization of whole cells and by solution and blot hybridization of subcellular fractions. Similar results were obtained both for wild-type cells and for a mutant containing amplified CAD genes in which the level of CAD RNA is 150-fold greater. CAD nuclear RNA is indistinguishable from mature mRNA by gel electrophoresis and blot hybridization. Discrete higher-molecular-weight precursors are undetectable, although the persistence of a short length of intervening sequence in the otherwise fully processed RNA is not excluded. CAD RNA is released from nuclei by sonication in physiological conditions in a ribonucleoprotein form that sediments as a broad peak at about 200S in a sucrose gradient. CAD sequences extracted from nuclei by treatment with EDTA and RNase are found in the 30S particles previously described.

A temperature sensitive mutant of a Chinese hamster cell line exhibiting high chromosomal breakage.

A temperature sensitive mutant exhibiting a very high level of chromosomal aberrations has been isolated from the Chinese hamster cell line E36. The chromosome aberrations, which include chromosome and chromatid breaks, multiradial configurations, dicentrics, and pulverizations, start to appear 1 h after a shift in temperature from 34 degrees C to 40.5 degrees C. The rate of sister chromatid exchange is not increased in this mutant. Analysis of somatic cell hybrids indicates that the mutation in this temperature sensitive mutant is dominant.