mRNA nuclear export.

The export of mRNA from the nucleus to the cytoplasm is an essential step in the expression of genetic information in eukaryotes. It is an energy-dependent process and involves transport across the nuclear pores. It requires both cis-acting ribonucleoprotein particle signals and specific trans-acting factors. Although much remains to be learned, recent information has begun to define this pathway at both the cellular and biochemical levels and indicates that it is used as a key regulatory step by several viruses.

mRNA export: travelling with DEAD box proteins.

Recent studies have shown that the putative RNA helicase protein UAP56 and its yeast homologue Sub2p are not only involved in pre-mRNA splicing but also required for the export of mRNA out of the nucleus, even if the mRNA is encoded by an intron-less gene.

The importin-beta family member Crm1p bridges the interaction between Rev and the nuclear pore complex during nuclear export.

BACKGROUND: The human immunodeficiency virus (HIV-1) uses the viral protein Rev to regulate gene expression by promoting the export of unspliced and partially spliced viral transcripts. Rev has been shown to function in a variety of organisms, including Saccharomyces cerevisiae. The export activity of Rev depends on a nuclear export signal (NES), which is believed to interact either directly or indirectly with the nuclear pore complex to carry out its export function. Crm1p is a member of the importin-beta protein family, other members of which are known to be directly involved in nuclear import. Crm1p has recently been shown to contribute to nuclear export in vertebrate systems. Here, we have studied this mechanism of nuclear to cytoplasmic transport. RESULTS: Viable mis-sense mutations in the CRM1 gene substantially reduced or eliminated the biological activity of Rev in S. cerevisiae, providing strong evidence that Crm1p also contributes to transport of Rev NES-containing proteins and ribonucleoproteins in this organism. Crm1p interacted with FG-repeat-containing nuclear pore proteins as well as Rev, and we have demonstrated that the previously described two-hybrid interaction between Rev and the yeast nuclear pore protein Rip1p is dependent on wild-type Crm1p. CONCLUSIONS: We conclude that Crm1p interacts with the Rev NES and nuclear pore proteins during delivery of cargo to the nuclear pore complex. Our findings also agree well with current experiments on Crm1p orthologs in Schizosaccharomyces pombe and in vertebrate systems.

U1 small nuclear ribonucleoprotein particle-protein interactions are revealed in Saccharomyces cerevisiae by in vivo competition assays.

Two highly conserved regions of the 586-nucleotide yeast (Saccharomyces cerevisiae) U1 small nuclear RNA (snRNA) can be mutated or deleted with little or no effect on growth rate: the universally conserved loop II (corresponding to the metazoan A loop) and the yeast core region (X. Liao, L. Kretzner, B. Séraphin, and M. Rosbash, Genes Dev. 4:1766-1774, 1990). To examine the contribution of these regions to U1 small nuclear ribonucleoprotein particle (snRNP) activity, a competitor U1 gene, encoding a nonfunctional U1 snRNA molecule, was introduced into a number of strains carrying a U1 snRNA gene with loop II or yeast core mutations. The presence of the nonfunctional U1 gene lowered the growth rate of these mutant strains but not wild-type strains, consistent with the notion that mutant U1 RNAs are less active than wild-type U1 snRNAs. A detailed analysis of the U1 snRNA levels and half-lives in a number of merodiploid strains suggests that these mutant U1 snRNAs interact with U1 snRNP proteins less well than do their wild-type counterparts. Competition for protein factors during snRNP assembly could account for a number of previous observations in both yeast and mammalian cells.

The yeast hnRNP-Like proteins Yra1p and Yra2p participate in mRNA export through interaction with Mex67p.

Yra1p is an essential nuclear protein which belongs to the evolutionarily conserved REF (RNA and export factor binding proteins) family of hnRNP-like proteins. Yra1p contributes to mRNA export in vivo and directly interacts with RNA and the shuttling mRNP export receptor Mex67p in vitro. Here we describe a second nonessential Saccharomyces cerevisiae family member, called Yra2p, which is able to complement a YRA1 deletion when overexpressed. Like other REF proteins, Yra1p and Yra2p consist of two highly conserved N- and C-terminal boxes and a central RNP-like RNA-binding domain (RBD). These conserved regions are separated by two more variable regions, N-vr and C-vr. Surprisingly, the deletion of a single conserved box or the deletion of the RBD in Yra1p does not affect viability. Consistently, neither the conserved N and C boxes nor the RBD is required for Mex67p and RNA binding in vitro. Instead, the N-vr and C-vr regions both interact with Mex67p and RNA. We further show that Yra1 deletion mutants which poorly interact with Mex67p in vitro affect the association of Mex67p with mRNP complexes in vivo and are paralleled by poly(A)(+) RNA export defects. These observations support the idea that Yra1p promotes mRNA export by facilitating the recruitment of Mex67p to the mRNP.

A role for nucleoporin FG repeat domains in export of human immunodeficiency virus type 1 Rev protein and RNA from the nucleus.

The human immunodeficiency virus type 1 Rev protein contains a nuclear export signal (NES) that is required for Rev-mediated RNA export in mammals as well as in the yeast Saccharomyces cerevisiae. The Rev NES has been shown to specifically interact with a human (hRIP/RAB1) and a yeast (yRip1p) protein in the two-hybrid assay. Both of these interacting proteins are related to FG nucleoporins on the basis of the presence of typical repeat motifs. This paper shows that Rev is able to interact with multiple FG repeat-containing nucleoporins from both S. cerevisiae and mammals; moreover, the ability of Rev NES mutants to interact with these FG nucleoporins parallels the ability of the mutants to promote RNA export in yeast and mammalian cells. The data also show that, after Xenopus oocyte nuclear injection, several FG nucleoporin repeat domains inhibit the export of both Rev protein and U small nuclear RNAs, suggesting that these nucleoporins participate in Rev-mediated and cellular RNA export. Interestingly, not all FG nucleoporin repeat domains produced the same pattern of RNA export inhibition. The results suggest that Rev and cellular mediators of RNA export can interact with multiple components of the nuclear pore complex during transport, analogous to the proposed mode of action of the nuclear protein import receptor.

Yeast Ran-binding protein 1 (Yrb1) shuttles between the nucleus and cytoplasm and is exported from the nucleus via a CRM1 (XPO1)-dependent pathway.

The RanGTP-binding protein RanBP1, which is located in the cytoplasm, has been implicated in release of nuclear export complexes from the cytoplasmic side of the nuclear pore complex. Here we show that Yrb1 (the yeast homolog of RanBP1) shuttles between the nucleus and the cytoplasm. Nuclear import of Yrb1 is a facilitated process that requires a short basic sequence within the Ran-binding domain (RBD). By contrast, nuclear export of Yrb1 requires an intact RBD, which forms a ternary complex with the Xpo1 (Crm1) NES receptor in the presence of RanGTP. Nuclear export of Yrb1, however, is insensitive towards leptomycin B, suggesting a novel type of substrate recognition between Yrb1 and Xpo1. Taken together, these data suggest that ongoing nuclear import and export is an important feature of Yrb1 function in vivo.

Relationship between sex hormone binding globulin and estrogen receptors in breast cancer.

It has been suggested that the level of sex hormone binding globulin (SHBG) is a better predictor of response of breast cancer to hormone treatment than the measurement of estrogen receptor (ER). However, no correlation of SHBG with ER status has been shown. To define the relationship between SHBG and the ER, the following study was undertaken. Fifty women with breast cancer and known ER status had SHBG measured by dextran-coated charcoal saturation analysis. The mean SHBG in all ER-positive patients was 20.7 +/- 2.4 (ng DHT bound/mL) and in all ER-negative patients was 11.5 +/- 2.0 (p less than 0.01). The mean premenopausal level of SHBG was 22.2 +/- 3.8 ng/mL and postmenopausal level was 20.0 +/- 5.7 ng/mL. There was no significant difference between these groups. ER-positive patients on tamoxifen had a SHBG of 29.8 +/- 9 ng/mL and ER-negative subjects on tamoxifen had a mean SHBG level of 8.3 +/- 3.4 ng/mL, p less than 0.01. ER-positive patients have a higher SHBG level than ER-negative patients; furthermore, this difference between ER-positive and ER-negative subjects was further demarcated by changes in SHBG after the subjects had been placed on tamoxifen.

Late relapse of testicular cancer.

The complete response (CR) rate of disseminated germ-cell tumors with current aggressive chemotherapy and surgical resection of localized residual disease is between 70%-80%. Most patients who relapse do so within the first year of therapy. We have observed seven patients with germ-cell tumors treated with a variety of modalities who relapsed after more than two years in CR (45-87 months). Five patients are alive after salvage therapy with follow-up too short to assess the durability of second remission. There were no features distinguishing late relapse patients from all patients treated with chemotherapy for germ-cell tumors. Follow-up in patients treated for germ-cell tumors with chemotherapy should be extended to five years before curability can be established.

Isolation and characterization of sarcomeric actin genes expressed in Xenopus laevis embryos.

A Xenopus laevis complementary DNA (cDNA) library prepared from messenger RNAs extracted from embryos has been screened for actin-coding sequences. Two cDNA clones corresponding to an alpha cardiac and an alpha skeletal muscle actin mRNA have been identified and characterized. From a genomic library, we have furthermore isolated the genes that correspond to the characterized cDNAs. In addition we have identified an actin processed gene which seems to be derived from a second type of skeletal muscle actin gene. Southern blot analysis of X. laevis DNA reveals that each of the three genes is present in at least two copies. In Xenopus tropicalis, a similar Southern blot analysis demonstrates that the three alpha actin genes exist as single copy. This result correlates with the genome duplication that has been proposed to have occurred recently in a X. laevis ancestor. A sequence comparison of the X. laevis cardiac and skeletal muscle actin cDNAs shows that the encoded peptides are highly conserved. Nevertheless, the numerous nucleotide changes at silent mutation sites suggest that the genes originated before the amphibia/reptile-bird divergence, more than 350 million years ago. Comparison of the promoters of the cardiac and skeletal actin genes, which are co-expressed in embryos, reveals a few common structural sequence elements.

A dynamic in vivo view of the HIV-I Rev-RRE interaction.

The export of pre-mRNAs coding for the structural genes of the human immunodeficiency virus type I depends on the interaction of the Rev protein with a highly structured viral RNA sequence, the Rev-responsive element (RRE). To gain information about the structure of the RRE and the determinants of the in vivo RRE-Rev interaction, we have analyzed the structure of the 351 nt RRE RNA within living yeast (Saccharomyces cerevisiae) by dimethyl sulfate probing with or without Rev. The in vivo structure in the absence of Rev is generally similar to the previously established solution structure. In addition, we observe a single hypermethylated guanine residue (G128), located within the Rev high-affinity binding site, in vitro as well as in vivo. The important homopurine interaction between residues 129 and 106 is required for the hyperreactivity, confirming its biological relevance. Expression of wild-type Rev leads to a protection of this region and to modifications of the RRE structure: the high-affinity site becomes further structured, and Stem IIA is destabilized. High-level expression of the oligomerization-defective mutant M4 protein leads to the same protections without destabilization of Stem IIA. Taken together with other observations, the data suggest that Rev captures the unusual conformation of the high-affinity site, followed by additional changes in the structure of the RRE.

A third striated muscle actin gene is expressed during early development in the amphibian Xenopus laevis.

During early embryonic development in the frog Xenopus laevis, several muscle-specific actin genes encoding distinct actin protein isoforms are activated in cells of the embryonic muscle. In addition to the cardiac (or alpha 1) and skeletal (or alpha 2) actin genes, a third muscle-specific actin gene is expressed in the same embryonic tissue. We have determined the complete nucleotide sequence of this third gene and examined its expression in embryonic and adult tissues. During embryogenesis, this femoral (alpha 3) actin gene is activated several hours later than its cardiac and skeletal counterparts and its transcripts are first detected after neurulation. The gene encodes a skeletal-type actin protein and is expressed exclusively in skeletal muscle in the adult frog. Two copies of this gene have been isolated from the tetraploid species Xenopus laevis, differing by only a few nucleotides in their protein-coding sequence. The related, diploid species, Xenopus tropicalis, possesses a single copy of the alpha 3 gene and its transcript is similarly conserved in nucleotide sequence. However, the X. tropicalis gene is expressed exclusively in embryonic stages of development. Comparison of the X. laevis and X. tropicalis alpha 3 gene promoters reveals extensive sequence homology, including several copies of a repeated motif that is common to other vertebrate striated-muscle actin gene promoters.

La proteins from Xenopus laevis. cDNA cloning and developmental expression.

In mammalian nuclei, newly-synthesized RNA polymerase III transcripts are transiently associated with a phosphorylated polypeptide of approximately 50 kDa called the La protein. Here we provide evidence that the frog Xenopus laevis contains mRNAs for two highly related La proteins, each apparently encoded by a single gene. Both forms of the La protein contain the RNP-80 motif previously identified in many RNA binding proteins. The steady state levels of La mRNAs and protein are approximately constant in oocytes, eggs and embryos. This implies a progressive and severe decrease in these levels on a per cell basis during early development. In particular, neither the La mRNA nor protein level increases at the mid-blastula transition, the time when RNA polymerase III transcription first occurs during embryogenesis.

Nuclear export of mRNA.

Export of mRNA through nuclear pore complexes (NPC) is preceded by multiple and well coordinated processing steps, resulting in the formation of an export competent ribonucleoprotein complex (mRNP). Numerous factors involved in the translocation of the mRNP through the NPC and its release into the cytoplasm have been isolated mainly through genetic approaches in yeast, and putative functional homologues have been identified in metazoan systems. Understanding the mechanism of mRNA export relies, in part, on the functional characterization of these factors and the establishment of a complete network of molecular interactions. Here we summarize recent progress in the characterization of yeast and mammalian components implicated in the export of an mRNA from the nucleus to the cytoplasm.

Indolent nonseminomatous germ cell tumor of testis. Prolonged survival of patient with persistent metastatic disease.

The complete response rate of disseminated nonseminomatous germ cell tumors (NSGCT) of the testes with current aggressive chemotherapy and surgical resection of residual disease is between 70 and 80 per cent. Those patients who do not attain complete response tend to have short survivals. A case is presented of a forty-one-year-old white man who has had nearly continuous evidence of metastatic embryonal carcinoma for more than eleven years. Although NSGCTs are characterized by rapid proliferation, early metastasis, high response rate to chemotherapy, and rapid death if uncontrolled, this case demonstrates an indolent form of disease with poor response to chemotherapy and yet prolonged survival in spite of uncontrolled disease. This is the first reported case of indolent metastatic germ cell neoplasm with survival of more than ten years.

Postoperative adjuvant chemotherapy and radiotherapy for cancer of the large bowel and rectum.

Forty patients with invasive regional-stage adenocarcinoma of the large bowel and rectum received adjuvant postoperative chemotherapy combined with doses of radiation below the maximal tissue tolerance level. This treatment was reserved for patients with stage B2, C1, and C2 lesions, with only two exceptions. The treatment was well tolerated. It appeared to result in a longer disease-free interval when compared with population-based results for patients with sigmoid cancer who had surgery alone. Our results paralleled those of the Gastrointestinal Tumor Study Group (GITSG) for combined adjuvant therapy of rectal cancer, who also indicated an advantage for long-term survival. Patients who received additional extended chemotherapy had at least the same percentage of favorable outcomes. Tumors above the peritoneal reflection also appeared to share the same improved results. We believe a multicenter randomized study should be performed to evaluate this group of patients.

Light source-induced error in computer-assisted image analysis with a video-based system.

OBJECTIVE: We tested the hypothesis that two different light sources, an alternating current fluorescent viewbox and a direct current halogen viewbox, do not differ with respect to their ability to illuminate reproducibly a radiograph during image capture. STUDY DESIGN: Two radiographs were taken: one with four hydroxyapatite chips mounted against a dry mandible and one without the chips. They were digitally subtracted with a video-based imaging system. The procedure was repeated at different times. RESULTS: A statistically significant difference among optical density measurements was found when the alternating current fluorescent viewbox (p < 0.001) was used and was related to light intensity variation. Such effect was not observed with the direct current halogen viewbox (p = 0.873). CONCLUSION: Study design efficiency was increased by 212% with the use of the direct current halogen viewbox so that to detect a specified treatment effect with a given level of statistical confidence, the sample size has to be 2.12 times greater if the alternating current fluorescent viewbox is used.