Nuclear pore complex clustering and nuclear accumulation of poly(A)+ RNA associated with mutation of the Saccharomyces cerevisiae RAT2/NUP120 gene.

To identify genes involved in the export of messenger RNA from the nucleus to the cytoplasm, we used an in situ hybridization assay to screen temperature-sensitive strains of Saccharomyces cerevisiae. This identified those which accumulated poly(A)+ RNA in their nuclei when shifted to the non-permissive temperature of 37 degrees C. We describe here the properties of yeast strains carrying mutations in the RAT2 gene (RAT - ribonucleic acid trafficking) and the cloning of the RAT2 gene. Only a low percentage of cells carrying the rat2-1 allele showed nuclear accumulation of poly(A)+ RNA when cultured at 15 degrees or 23 degrees C, but within 4 h of a shift to the nonpermissive temperature of 37 degrees C, poly(A)+ RNA accumulated within the nuclei of approximately 80% of cells. No defect was seen in the nuclear import of a reporter protein bearing a nuclear localization signal. Nuclear pore complexes (NPCs) are distributed relatively evenly around the nuclear envelope in wild-type cells. In cells carrying either the rat2-1 or rat2-2 allele, NPCs were clustered together into one or a few regions of the nuclear envelope. This clustering was a constitutive property of mutant cells. NPCs remained clustered in crude nuclei isolated from mutant cells, indicating that these clusters are not able to redistribute around the nuclear envelope when nuclei are separated from cytoplasmic components. Electron microscopy revealed that these clusters were frequently found in a protuberance of the nuclear envelope and were often located close to the spindle pole body. The RAT2 gene encodes a 120-kD protein without similarity to other known proteins. It was essential for growth only at 37 degrees C, but the growth defect at high temperature could be suppressed by growth of mutant cells in the presence of high osmolarity media containing 1.0 M sorbitol or 0.9 M NaCl. The phenotypes seen in cells carrying a disruption of the RAT2 gene were very similar to those seen with the rat2-1 and rat2-2 alleles. Epitope tagging was used to show that Rat2p is located at the nuclear periphery and co-localizes with yeast NPC proteins recognized by the RL1 monoclonal antibody. The rat2-1 allele was synthetically lethal with both the rat3-1/nup133-1 and rat7-1/nup159-1 alleles. These results indicate that the product of this gene is a nucleoporin which we refer to as Rat2p/Nup120p.

In vitro initiation of transcription by RNA polymerase II on in vivo-assembled chromatin templates.

We have studied the initiation of transcription in vitro by RNA polymerase II on simian virus 40 (SV40) minichromosomal templates isolated from infected cells. The efficiency and pattern of transcription from the chromatin templates were compared with those from viral DNA templates by using two in vitro transcription systems, either HeLa whole-cell extract or basal transcription factors, RNA polymerase II, and one of two SV40 promoter-binding transcription factors, LSF and Sp1. Dramatic increases in numbers of transcripts upon addition of transcription extract and different patterns of usage of the multiple SV40 initiation sites upon addition of Sp1 versus LSF strongly suggested that transcripts were being initiated from the minichromosomal templates in vitro. That the majority of transcripts from the minichromosomes were due to initiation de novo was demonstrated by the efficient transcription observed in the presence of alpha-amanitin, which inhibited minichromosome-associated RNA polymerase II, and an alpha-amanitin-resistant RNA polymerase II, which initiated transcription in vitro. The pattern of transcription from the SV40 late and early promoters on the minichromosomal templates was similar to the in vivo pattern of transcription during the late stages of viral infection and was distinct from the pattern of transcription generated from viral DNA in vitro. In particular, the late promoter of the minichromosomal templates was transcribed with high efficiency, similar to viral DNA templates, while the early-early promoter of the minichromosomal templates was inhibited 10- to 15-fold. Finally, the number of minichromosomes competent to initiate transcription in vitro exceeded the amount actively being transcribed in vivo.

C-terminal truncations of the yeast nucleoporin Nup145p produce a rapid temperature-conditional mRNA export defect and alterations to nuclear structure.

A screen for temperature-sensitive mutants of Saccharomyces cerevisiae defective in nucleocytoplasmic trafficking of poly(A)+ RNA has identified an allele of the NUP145 gene, which encodes an essential nucleoporin. NUP145 was previously identified by using a genetic synthetic lethal screen (E. Fabre, W. C. Boelens, C. Wimmer, I. W. Mattaj, and E. C. Hurt, Cell 78:275-289, 1994) and by using a monoclonal antibody which recognizes the GLFG family of vertebrate and yeast nucleoporins (S. R. Wente and G. Blobel, J. Cell Biol. 125:955-969, 1994). Cells carrying the new allele, nup145-10, grew at 23 and 30 degrees C but were unable to grow at 37 degrees C. Many cells displayed a modest accumulation of poly(A)+ RNA under permissive growth conditions, and all cells showed dramatic and rapid nuclear accumulation of poly(A)+ RNA following a shift to 37 degrees C. The mutant allele contains a nonsense codon which truncates the 1,317-amino-acid protein to 698 amino acids. This prompted us to examine the role of the carboxyl half of Nup145p. Several additional alleles that encode C-terminally truncated proteins or proteins containing internal deletions of portions of the carboxyl half of Nup145p were constructed. Analysis of these mutants indicates that some sequences between amino acids 698 and 1095 are essential for RNA export and for growth at 37 degrees C. In these strains, nuclear accumulation of poly(A)+ RNA and fragmentation of the nucleolus occurred rapidly following a shift to 37 degrees C. Constitutive defects in nuclear pore complex distribution and nuclear structure were also seen in these strains. Although cells lacking Nup145p grew extremely slowly at 23 degrees C and did not grow at 30 degrees C, efficient growth at 23 or 30 degrees C occurred as long as cells produced either the amino 58% or the carboxyl 53% of Nup145p. Strains carrying alleles of NUP145 lacking up to 200 amino acids from the carboxy terminus were viable at 37 degrees C but displayed nucleolar fragmentation and some nuclear accumulation of poly(A)+ RNA following a shift to 37 degrees C. Surprisingly, these strains grew efficiently at 37 degrees C in spite of a reduction in the level of synthesis of rRNAs to approximately 25% of the wild-type level.

Pleiotropic nuclear defects associated with a conditional allele of the novel nucleoporin Rat9p/Nup85p.

In a screen for mutants defective in nucleocytoplasmic export of mRNA, we have identified a new component of the Saccharomyces cerevisiae nuclear pore complex (NPC). The RAT9/NUP85 (ribonucleic acid trafficking) gene encodes an 84.9-kDa protein that we have localized to NPCs by tagging the RAT9/NUP85 gene with the in vivo molecular marker Green Fluorescent Protein. In cells containing either the rat9-1 allele or a complete deletion of the RAT9/NUP85 gene, poly(A)+ RNA accumulates rapidly in nuclei after a shift from 23 degrees C to 37 degrees C. Under these same conditions, rapid fragmentation of the nucleolus was also observed. At the permissive growth temperature in rat9-1 or RAT9 deletion strains, the nuclear envelope (NE) becomes detached from the main body of the nucleus, forming long thin double sheets of NE. NPCs within these sheets are clustered and some appear to be locked together between opposing sheets of NE such that their nucleoplasmic faces are in contact. The Rat9/Nup85 protein could not be detected in cells carrying a mutation of RAT2/NUP120, suggesting that Rat9p/Nup85p cannot be assembled into NPCs in the absence of Rat2p/Nup120p. In contrast,Rat9/ Nup85 protein was readily incorporated into NPCs in strains carrying mutant alleles of other nucleoporin genes. The possible role of Rat9p/Nup85p in NE integrity and the loss of nucleoporins when another nucleoporin is mutant or absent are discussed.

Mutation or deletion of the Saccharomyces cerevisiae RAT3/NUP133 gene causes temperature-dependent nuclear accumulation of poly(A)+ RNA and constitutive clustering of nuclear pore complexes.

To identify genes whose products play potential roles in the nucleocytoplasmic export of messenger RNA, we isolated temperature-sensitive strains of Saccharomyces cerevisiae and examined them by fluorescent in situ hybridization. With the use of a digoxigen-tagged oligo-(dT)50 probe, we identified those that showed nuclear accumulation of poly(A)+ RNA when cells were shifted to the nonpermissive temperature. We describe here the properties of yeast strains bearing the rat3-1 mutation (RAT-ribonucleic acid trafficking) and the cloning of the RAT3 gene. When cultured at the permissive temperature of 23 degrees C, fewer than 10% of cells carrying the rat3-1 allele showed nuclear accumulation of poly(A)+ RNA, whereas approximately 70% showed nuclear accumulation of poly(A)+ RNA, whereas approximately 70% showed nuclear accumulation of poly(A)+ RNA after a shift to 37 degrees C for 4 h. In wild-type cells, nuclear pore complexes (NPCs) are distributed relatively evenly around the nuclear envelope. Both indirect immunofluorescence analysis and electron microscopy of rat3-1 cells indicated that NPCs were clustered into one or a few regions of the NE in mutant cells. Similar NPC clustering was seen in mutant cells cultured at temperatures between 15 degrees C and 37 degrees C. The RAT3 gene encodes an 1157-amino acid protein without similarity to other known proteins. It is essential for growth only at 37 degrees C. Cells carrying a disruption of the RAT3 gene were very similar to cells carrying the original rat3-1 mutation; they showed temperature-dependent nuclear accumulation of poly(A)+ RNA and exhibited constitutive clustering of NPCs. Epitope tagging of Rat3p demonstrated that it is located at the nuclear periphery and co-localizes with nuclear pore proteins recognized by the RL1 monoclonal antibody. We refer to this nucleoporin as Rat3p/Nup133p.

Measurement of thyroid-stimulating hormone in human amniotic fluid.

TSH was measured in human amniotic fluid after 3- to 8-fold concentration of the fluids. Amniotic fluid TSH was identical to standard pituitary TSH by immunological and gel chromatographic criteria. In 201 samples of normal second trimester amniotic fluid (16-19 weeks of pregnancy), amniotic fluid TSH concentrations had a mean value of 0.4 microunits/ml (range, less than 0.15 to 1.7 microunits/ml). In 21 samples of third trimester amniotic fluid (obtained to check fetal lung maturity), amniotic fluid TSH concentrations had a mean value of 0.25 microunits/ml (range, less than 0.15 to 0.55 microunits/ml). The capability of measuring TSH in amniotic fluid and the relative constancy of these values between the second and third trimesters of pregnancy suggest that the determination of TSH levels in amniotic fluid may be useful in the diagnosis of fetal hypothyroidism in utero.

Adenovirus helper function activity of simian virus 40 T antigen mutants.

The SV40 large T antigen provides a helper function that permits human adenovirus yields in monkey cells to approach those obtained in human cells. The carboxy-terminus of large T antigen is involved in providing this activity. The ability of a large number of SV40 mutants affecting T antigen to enhance the growth of adenovirus type 2 in the CV-1 line of African green monkey kidney cells was examined. Mutation of those serines and threonines at the carboxy terminus which are normally phosphorylated had no effect on adenovirus helper function. A cytoplasmic T antigen was very effective in providing adenovirus helper function. Mutants that produce unstable T antigens provided helper function, but to a reduced degree. Finally, mutations in T antigen which permit it to interfere trans-dominantly with replication catalyzed by wildtype T antigen provided adenovirus helper function at wildtype levels.

Spatial constraints on polyadenylation signal function.

Efficient cleavage and polyadenylation of eukaryotic messenger RNAs require at least two signal elements: an AAUAAA or closely related sequence located 7-30 base pairs (bp) upstream of the site of processing, and a G/U- or U-rich sequence located 3' to the cleavage site. The herpes simplex virus type 1 thymidine kinase (tk) gene contains two copies of the AATAAA hexanucleotide and a GT-rich region. We have shown that the first AATAAA and the GT-rich region are essential for efficient processing, both in vivo and in vitro, whereas the second AATAAA does not appear to play any role in the formation of tk mRNA 3' ends. The failure of a signal containing only the second AATAAA and the GT-rich element to signal cleavage and polyadenylation suggested that these two elements might be too close together to constitute a functional polyadenylation signal. The experiments described in this report were directed at determining the effects on mRNA 3' end formation of alterations in spacing between signal elements. Wild-type tk contains 19 bp between these two elements. Constructs were made in which an AATAAA and the GT-rich region were separated by various distances ranging from 7 to 43 bp. The quantity and location of 3' ends of the tk mRNA produced by these constructs in Cos-1 cells were measured by S1 nuclease protection analysis. Signal efficiency was gradually reduced as the separation between the two signal elements was increased; with a separation of 43 bp, the signal functioned at approximately one-eighth the efficiency of the parental construction. Bringing the two signals closer together resulted in decreased signal efficiency; with a separation of 7 or 9 bp, no tk mRNA polyadenylated within the normal region was produced. Altering the sequences between these two elements without changing the distance had small effects on processing efficiency.

Yeast heat shock mRNAs are exported through a distinct pathway defined by Rip1p.

We reported previously that heat or ethanol shock in Saccharomyces cerevisiae leads to nuclear retention of most poly(A)+ RNA but heat shock mRNAs (encoding Hsp70 proteins Ssa1p and Ssa4p) are efficiently exported in a process that is independent of the small GTPase Ran/Gsp1p, which is essential for most nucleocytoplasmic transport. To gain further insights into proteins essential or nonessential for export of heat shock mRNAs, in situ hybridization analyses to detect mRNA and pulse-labeling of proteins were used to examine several yeast mutant strains for their ability to export heat shock mRNAs following stress. Rip1p is a 42-kD protein associated with nuclear pore complexes and contains nucleoporin-like repeat sequences. It is dispensable for growth of yeast cells under normal conditions, but we report that it is essential for the export of heat shock mRNAs following stress. When SSA4 mRNA was induced from a GAL promoter in the absence of stress, it was efficiently exported in a strain lacking RIP1, indicating that Rip1p is required for export of heat shock mRNAs only following stress. Npl3p, a key mediator of export of poly(A)+ RNA, was not required for heat shock mRNA export, whereas Rss1p/Gle1p, a NES-containing factor essential for poly(A)+ RNA export, was also required for export of heat shock mRNAs after stress. High-level expression of the HIV-1 Rev protein, but not of Rev mutants, led to a partial block in export of heat shock mRNAs following stress. The data suggest a model wherein the requirement for Npl3p defines the mRNA export pathway, the requirement for Rip1p defines a pathway used for export of heat shock mRNAs after stress, and additional factors, including Rss1p/Gle1p and several nucleoporins (Rat7p/Nup159p, Rat2p/Nup120p, and Nup145p/Rat10p), are required in both pathways.