soc-2 encodes a leucine-rich repeat protein implicated in fibroblast growth factor receptor signaling

Activation of fibroblast growth factor (FGF) receptors elicits diverse cellular responses including growth, mitogenesis, migration, and differentiation. The intracellular signaling pathways that mediate these important processes are not well understood. In Caenorhabditis elegans, suppressors of clr-1 identify genes, termed soc genes, that potentially mediate or activate signaling through the EGL-15 FGF receptor. We demonstrate that three soc genes, soc-1, soc-2, and sem-5, suppress the activity of an activated form of the EGL-15 FGF receptor, consistent with the soc genes functioning downstream of EGL-15. We show that soc-2 encodes a protein composed almost entirely of leucine-rich repeats, a domain implicated in protein–protein interactions. We identified a putative human homolog, SHOC-2, which is 54% identical to SOC-2. We find that shoc-2 maps to 10q25, shoc-2 mRNA is expressed in all tissues assayed, and SHOC-2 protein is cytoplasmically localized. Within the leucine-rich repeats of both SOC-2 and SHOC-2 are two YXNX motifs that are potential tyrosine-phosphorylated docking sites for the SEM-5/GRB2 Src homology 2 domain. However, phosphorylation of these residues is not required for SOC-2 function in vivo, and SHOC-2 is not observed to be tyrosine phosphorylated in response to FGF stimulation. We conclude that this genetic system has allowed for the identification of a conserved gene implicated in mediating FGF receptor signaling in C. elegans.

DsrA RNA regulates translation of RpoS message by an anti-antisense mechanism, independent of its action as an antisilencer of transcription

DsrA RNA regulates both transcription, by overcoming transcriptional silencing by the nucleoid-associated H-NS protein, and translation, by promoting efficient translation of the stress σ factor, RpoS. These two activities of DsrA can be separated by mutation: the first of three stem-loops of the 85 nucleotide RNA is necessary for RpoS translation but not for anti-H-NS action, while the second stem-loop is essential for antisilencing and less critical for RpoS translation. The third stem-loop, which behaves as a transcription terminator, can be substituted by the trp transcription terminator without loss of either DsrA function. The sequence of the first stem-loop of DsrA is complementary with the upstream leader portion of rpoS messenger RNA, suggesting that pairing of DsrA with the rpoS message might be important for translational regulation. Mutations in the Rpos leader and compensating mutations in DsrA confirm that this predicted pairing is necessary for DsrA stimulation of RpoS translation. We propose that DsrA pairing stimulates RpoS translation by acting as an anti-antisense RNA, freeing the translation initiation region from the cis-acting antisense RNA and allowing increased translation.

A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2

Human cytomegalovirus (CMV), a herpesvirus that causes congenital disease and opportunistic infections in immunocompromised individuals, encodes functions that facilitate efficient viral propagation by altering host cell behavior. Here we show that CMV blocks apoptosis mediated by death receptors and encodes a mitochondria-localized inhibitor of apoptosis, denoted vMIA, capable of suppressing apoptosis induced by diverse stimuli. vMIA, a product of the viral UL37 gene, inhibits Fas-mediated apoptosis at a point downstream of caspase-8 activation and Bid cleavage but upstream of cytochrome c release, while residing in mitochondria and associating with adenine nucleotide translocator. These functional properties resemble those ascribed to Bcl-2; however, the absence of sequence similarity to Bcl-2 or any other known cell death suppressors suggests that vMIA defines a previously undescribed class of anti-apoptotic proteins.

The importin/karyopherin Kap114 mediates the nuclear import of TATA-binding protein

Two high copy suppressors of temperature-sensitive TATA-binding protein (TBP) mutants were isolated. One suppressor was TIF51A, which encodes eukaryotic translation initiation factor 5A. The other high copy suppressor, YGL241W, also known as KAP114, is one of 14 importin/karyopherin proteins in yeast. These proteins mediate the transport of specific macromolecules into and out of the nucleus. Cells lacking Kap114 partially mislocalize TBP to the cytoplasm. Kap114 binds TBP in vitro, and binding is disrupted in the presence of GTPγS. Therefore, Kap114 is an importer of TBP into the nucleus, but alternative import pathways must also exist.

SMRT corepressor interacts with PLZF and with the PML-retinoic acid receptor α (RARα) and PLZF-RARα oncoproteins associated with acute promyelocytic leukemia

Retinoic acid receptors (RARs) are hormone-regulated transcription factors that control key aspects of normal differentiation. Aberrant RAR activity may be a causal factor in neoplasia. Human acute promyelocytic leukemia, for example, is tightly linked to chromosomal translocations that fuse novel amino acid sequences (denoted PML, PLZF, and NPM) to the DNA-binding and hormone-binding domains of RARα. The resulting chimeric receptors have unique transcriptional properties that may contribute to leukemogenesis. Normal RARs repress gene transcription by associating with ancillary factors denoted corepressors (also referred to as SMRT, N-CoR, TRAC, or RIP13). We report here that the PML-RARα and PLZF-RARα oncoproteins retain the ability of RARα to associate with corepressors, and that this corepressor association correlates with certain aspects of the leukemic phenotype. Unexpectedly, the PLZF moiety itself can interact with SMRT corepressor. This interaction with corepressor is mediated, in part, by a POZ motif within PLZF. Given the presence of POZ motifs in a number of known transcriptional repressors, similar interactions with SMRT may play a role in transcriptional silencing by a variety of both receptor and nonreceptor transcription factors.

Evidence for the recent horizontal transfer of long terminal repeat retrotransposon

The evolutionary dynamics existing between transposable elements (TEs) and their host genomes have been likened to an “arms race.” The selfish drive of TEs to replicate, in turn, elicits the evolution of host-mediated regulatory mechanisms aimed at repressing transpositional activity. It has been postulated that horizontal (cross-species) transfer may be one effective strategy by which TEs and other selfish genes can escape host-mediated silencing mechanisms over evolutionary time; however, to date, the most definitive evidence that TEs horizontally transfer between species has been limited to class II or DNA-type elements. Evidence that the more numerous and widely distributed retroelements may also be horizontally transferred between species has been more ambiguous. In this paper, we report definitive evidence for a recent horizontal transfer of the copia long terminal repeat retrotransposon between Drosophila melanogaster and Drosophila willistoni.

Changes of telomere length cause reciprocal changes in the lifespan of mother cells in Saccharomyces cerevisiae

Budding yeast cells divide asymmetrically, giving rise to a mother and its daughter. Mother cells have a limited division potential, called their lifespan, which ends in proliferation-arrest and lysis. In this report we mutate telomerase in Saccharomyces cerevisiae to shorten telomeres and show that, rather than shortening lifespan, this leads to a significant extension in lifespan. This extension requires the product of the SIR3 gene, an essential component of the silencing machinery which binds to telomeres. In contrast, longer telomeres in a genotypically wild-type strain lead to a decrease in lifespan. These findings suggest that the length of telomeres dictates the lifespan by regulating the amount of the silencing machinery available to nontelomeric locations in the yeast genome.

Specific atomic groups and RNA helix geometry in acceptor stem recognition by a tRNA synthetase

Oligonucleotides that recapitulate the acceptor stems of tRNAs are substrates for aminoacylation by many tRNA synthetases in vitro, even though these substrates are missing the anticodon trinucleotides of the genetic code. In the case of tRNAAla a single acceptor stem G⋅U base pair at position 3·70 is essential, based on experiments where the wobble pair has been replaced by alternatives such as I⋅U, G⋅C, and A⋅U, among others. These experiments led to the conclusion that the minor-groove free 2-amino group (of guanosine) of the G⋅U wobble pair is essential for charging. Moreover, alanine-inserting tRNAs (amber suppressors) that replace G⋅U with mismatches such as G⋅A and C⋅A are partially active in vivo and can support growth of an Escherichia coli tRNAAla knockout strain, leading to the hypothesis that a helix irregularity and nucleotide functionalities are important for recognition. Herein we investigate the charging in vitro of oligonucleotide and full-length tRNA substrates that contain mismatches at the position of the G⋅U pair. Although most of these substrates have undetectable activity, G⋅A and C⋅A variants retain some activity, which is, nevertheless, reduced by at least 100-fold. Thus, the in vivo assays are much less sensitive to large changes in aminoacylation kinetic efficiency of 3·70 variants than is the in vitro assay system. Although these functional data do not clarify all of the details, it is now clear that specific atomic groups are substantially more important in determining kinetic efficiency than is a helical distortion. By implication, the activity of mutant tRNAs measured in the in vivo assays appears to be more dependent on factors other than aminoacylation kinetic efficiency.

Structure of the imprinted mouse Snrpn gene and establishment of its parental-specific methylation pattern

The mouse Snrpn gene encodes the Smn protein, which is involved in RNA splicing. The gene maps to a region in the central part of chromosome 7 that is syntenic to the Prader–Willi/Angelman syndromes (PWS-AS) region on human chromosome 15q11-q13. The mouse gene, like its human counterpart, is imprinted and paternally expressed, primarily in brain and heart. We provide here a detailed description of the structural features and differential methylation pattern of the gene. We have identified a maternally methylated region at the 5′ end (DMR1), which correlates inversely with the Snrpn paternal expression. We also describe a region at the 3′ end of the gene (DMR2) that is preferentially methylated on the paternal allele. Analysis of Snrpn mRNA levels in a methylase-deficient mouse embryo revealed that maternal methylation of DMR1 may play a role in silencing the maternal allele. Yet both regions, DMR1 and DMR2, inherit the parental-specific methylation profile from the gametes. This methylation pattern is erased in 12.5-days postcoitum (dpc) primordial germ cells and reestablished during gametogenesis. DMR1 is remethylated during oogenesis, whereas DMR2 is remethylated during spermatogenesis. Once established, these methylation patterns are transmitted to the embryo and maintained, protected from methylation changes during embryogenesis and cell differentiation. Transfections of DMR1 and DMR2 into embryonic stem cells and injection into pronuclei of fertilized eggs reveal that embryonic cells lack the capacity to establish anew the differential methylation pattern of Snrpn. That all PWS patients lack DMR1, together with the overall high resemblance of the mouse gene to the human SNRPN, offers an excellent experimental tool to study the regional control of this imprinted chromosomal domain.

Corepressor SMRT binds the BTB/POZ repressing domain of the LAZ3/BCL6 oncoprotein

The LAZ3/BCL6 (lymphoma-associated zinc finger 3/B cell lymphomas 6) gene frequently is altered in non-Hodgkin lymphomas. It encodes a sequence-specific DNA binding transcriptional repressor that contains a conserved N-terminal domain, termed BTB/POZ (bric-à-brac tramtrack broad complex/pox viruses and zinc fingers). Using a yeast two-hybrid screen, we show here that the LAZ3/BCL6 BTB/POZ domain interacts with the SMRT (silencing mediator of retinoid and thyroid receptor) protein. SMRT originally was identified as a corepressor of unliganded retinoic acid and thyroid receptors and forms a repressive complex with a mammalian homolog of the yeast transcriptional repressor SIN3 and the HDAC-1 histone deacetylase. Protein binding assays demonstrate that the LAZ3/BCL6 BTB/POZ domain directly interacts with SMRT in vitro. Furthermore, DNA-bound LAZ3/BCL6 recruits SMRT in vivo, and both overexpressed proteins completely colocalize in nuclear dots. Finally, overexpression of SMRT enhances the LAZ3/BCL6-mediated repression. These results define SMRT as a corepressor of LAZ3/BCL6 and suggest that LAZ3/BCL6 and nuclear hormone receptors repress transcription through shared mechanisms involving SMRT recruitment and histone deacetylation.

Oncogenic transformation induced by the Qin protein is correlated with transcriptional repression

The retroviral oncogene qin codes for a protein that belongs to the family of the winged helix transcription factors. The viral Qin protein, v-Qin, differs from its cellular counterpart, c-Qin, by functioning as a stronger transcriptional repressor and a more efficient inducer of tumors. This observation suggests that repression may be important in tumorigenesis. To test this possibility, chimeric proteins were constructed in which the Qin DNA-binding domain was fused to either a strong repressor domain (derived from the Drosophila Engrailed protein) or a strong activator domain (from the herpes simplex virus VP16 protein). The chimeric transcriptional repressor, Qin–Engrailed, transformed chicken embryo fibroblasts in culture and induced sarcomas in young chickens. The chimeric activator, Qin–VP16, failed to transform cells in vitro or in vivo and caused cellular resistance to oncogenic transformation by Qin. These data support the conclusion that the Qin protein induces oncogenic transformation by repressing the transcription of genes which function as negative growth regulators or tumor suppressors.

BRCA1 is associated with the centrosome during mitosis

Centrosomes and their associated microtubules direct events during mitosis and control the organization of animal cell structures and movement during interphase. The centrosome replicates during the cell cycle, directs the assembly of bipolar mitotic spindles, and plays an important role in maintaining the fidelity of cell division. Recently, tumor suppressors such as p53 and retinoblastoma protein pRB have been localized to the centrosome in a cell cycle-dependent manner. Immunofluorescence microscopy and analysis of isolated centrosomes now provide evidence that BRCA1 protein, a suppressor of tumorigenesis in breast and ovary, also is associated with centrosomes during mitosis. Our results indicate that BRCA1 localizes with the centrosome during mitosis and coimmunoprecipitates with γ-tubulin, a centrosomal component essential for nucleation of microtubules. Furthermore, γ-tubulin associates preferentially with a hypophosphorylated form of BRCA1.

Mso1p: A yeast protein that functions in secretion and interacts physically and genetically with Sec1p

The yeast Sec1p protein functions in the docking of secretory transport vesicles to the plasma membrane. We previously have cloned two yeast genes encoding syntaxins, SSO1 and SSO2, as suppressors of the temperature-sensitive sec1–1 mutation. We now describe a third suppressor of sec1–1, which we call MSO1. Unlike SSO1 and SSO2, MSO1 is specific for sec1 and does not suppress mutations in any other SEC genes. MSO1 encodes a small hydrophilic protein that is enriched in a microsomal membrane fraction. Cells that lack MSO1 are viable, but they accumulate secretory vesicles in the bud, indicating that the terminal step in secretion is partially impaired. Moreover, loss of MSO1 shows synthetic lethality with mutations in SEC1, SEC2, and SEC4, and other synthetic phenotypes with mutations in several other late-acting SEC genes. We further found that Mso1p interacts with Sec1p both in vitro and in the two-hybrid system. These findings suggest that Mso1p is a component of the secretory vesicle docking complex whose function is closely associated with that of Sec1p.

Neural restrictive silencer factor recruits mSin3 and histone deacetylase complex to repress neuron-specific target genes

Accumulative evidence suggests that more than 20 neuron-specific genes are regulated by a transcriptional cis-regulatory element known as the neural restrictive silencer (NRS). A trans-acting repressor that binds the NRS, NRSF [also designated RE1-silencing transcription factor (REST)] has been cloned, but the mechanism by which it represses transcription is unknown. Here we show evidence that NRSF represses transcription of its target genes by recruiting mSin3 and histone deacetylase. Transfection experiments using a series of NRSF deletion constructs revealed the presence of two repression domains, RD-1 and RD-2, within the N- and C-terminal regions, respectively. A yeast two-hybrid screen using the RD-1 region as a bait identified a short form of mSin3B. In vitro pull-down assays and in vivo immunoprecipitation-Western analyses revealed a specific interaction between NRSF-RD1 and mSin3 PAH1-PAH2 domains. Furthermore, NRSF and mSin3 formed a complex with histone deacetylase 1, suggesting that NRSF-mediated repression involves histone deacetylation. When the deacetylation of histones was inhibited by tricostatin A in non-neuronal cells, mRNAs encoding several neuronal-specific genes such as SCG10, NMDAR1, and choline acetyltransferase became detectable. These results indicate that NRSF recruits mSin3 and histone deacetylase 1 to silence neural-specific genes and suggest further that repression of histone deacetylation is crucial for transcriptional activation of neural-specific genes during neuronal terminal differentiation.

Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm

Under physiological conditions, the Escherichia coli cytoplasm is maintained in a reduced state that strongly disfavors the formation of stable disulfide bonds in proteins. However, mutants in which the reduction of both thioredoxins and glutathione is impaired (trxB gor mutants) accumulate oxidized, enzymatically active alkaline phosphatase in the cytoplasm. These mutants grow very poorly in the absence of an exogenous reductant and accumulate extragenic suppressors at a high frequency. One such suppressor strain, FA113, grows almost as rapidly as the wild type in the absence of reductant, exhibits slightly faster kinetics of disulfide bond formation, and has fully induced activity of the transcriptional activator, OxyR. FA113 gave substantially higher yields of properly oxidized proteins compared with wild-type or trxB mutant strains. For polypeptides with very complex patterns of disulfide bonds, such as vtPA and the full-length tPA, the amount of active protein was further enhanced up to 15-fold by co-expression of TrxA (thioredoxin 1) mutants with different redox potentials, or 20-fold by the protein disulfide isomerase, DsbC. Remarkably, higher yields of oxidized, biologically active proteins were obtained by expression in the cytoplasm of E. coli FA113 compared with what could be achieved via secretion into the periplasm of a wild-type strain, even under optimized conditions. These results demonstrate that the cytoplasm can be rendered sufficiently oxidizing to allow efficient formation of native disulfide bonds without compromising cell viability.