Cloning of mDEAH9, a putative RNA helicase and mammalian homologue of Saccharomyces cerevisiae splicing factor Prp43

Yeast splicing factor Prp43, a DEAH box protein of the putative RNA helicase/RNA-dependent NTPase family, is a splicing factor that functions late in the pre-mRNA splicing pathway to facilitate spliceosome disassembly. In this paper we report cDNA cloning and characterization of mDEAH9, an apparent mammalian homologue of Prp43. Amino acid sequence comparison revealed that the two proteins are ≈65% identical over a 500-aa region spanning the central helicase domain and the C-terminal region. Expression of mDEAH9 in S. cerevisiae bearing a temperature-sensitive mutation in prp43 was sufficient to restore growth at the nonpermissive temperature. This functional complementation was specific, as mouse mDEAH9 failed to complement mutations in related splicing factor genes prp16 or prp22. Finally, double label immunofluorescence experiments performed with mammalian cells revealed colocalization of mDEAH9 and splicing factor SC35 in punctate nuclear speckles. Thus, the hypothesis that mDEAH9 represents the mammalian homologue of yeast Prp43 is supported by its high sequence homology, functional complementation, and colocalization with a known splicing factor in the nucleus. Our results provide additional support for the hypothesis that the spliceosomal machinery that mediates regulated, dynamic changes in conformation of pre-mRNA and snRNP RNAs has been highly conserved through evolution.

Incomplete penetrance of familial retinoblastoma linked to germ-line mutations that result in partial loss of RB function

To study the molecular basis for the clinical phenotype of incomplete penetrance of familial retinoblastoma, we have examined the functional properties of three RB mutations identified in the germ line of five different families with low penetrance. RB mutants isolated from common adult cancers and from classic familial retinoblastoma (designated as classic RB mutations) are unstable and generally do not localize to the nucleus, do not undergo cyclin-dependent kinase (cdk)-mediated hyperphosphorylation, show absent protein “pocket” binding activity, and do not suppress colony growth of RB(−) cells. In contrast, two low-penetrant alleles (661W and “deletion of codon 480”) retained the ability to localize to the nucleus, showed normal cdk-mediated hyperphosphorylation in vivo, exhibited a binding pattern to simian virus 40 large T antigen using a quantitative yeast two-hybrid assay that was intermediate between classic mutants (null) and wild-type RB, and had absent E2F1 binding in vitro. A third, low-penetrant allele, “deletion of RB exon 4,” showed minimal hyperphosphorylation in vivo but demonstrated detectable E2F1 binding in vitro. In addition, each low-penetrant RB mutant retained the ability to suppress colony growth of RB(−) tumor cells. These findings suggest two categories of mutant, low-penetrant RB alleles. Class 1 alleles correspond to promoter mutations, which are believed to result in reduced or deregulated levels of wild-type RB protein, whereas class 2 alleles result in mutant proteins that retain partial activity. Characterization of the different subtypes of class 2 low-penetrant genes may help to define more precisely functional domains within the RB product required for tumor suppression.

Pathogenic implications of mutations in the tau gene in pallido-ponto-nigral degeneration and related neurodegenerative disorders linked to chromosome 17

Pallido-ponto-nigral degeneration (PPND) is one of the most well characterized familial neurodegenerative disorders linked to chromosome 17q21–22. These hereditary disorders are known collectively as frontotemporal dementia (FTD) and parkinsonism linked to chromosome 17 (FTDP-17). Although the clinical features and associated regional variations in the neuronal loss observed in different FTDP-17 kindreds are diverse, the diagnostic lesions of FTDP-17 brains are tau-rich filaments in the cytoplasm of specific subpopulations of neurons and glial cells. The microtubule associated protein (tau) gene is located on chromosome 17q21–22. For these reasons, we investigated the possibility that PPND and other FTDP-17 syndromes might be caused by mutations in the tau gene. Two missense mutations in exon 10 of the tau gene that segregate with disease, Asn279Lys in the PPND kindred and Pro301Leu in four other FTDP-17 kindreds, were found. A third mutation was found in the intron adjacent to the 3′ splice site of exon 10 in patients from another FTDP-17 family. Transcripts that contain exon 10 encode tau isoforms with four microtubule (MT)-binding repeats (4Rtau) as opposed to tau isoforms with three MT-binding repeats (3Rtau). The insoluble tau aggregates isolated from brains of patients with each mutation were analyzed by immunoblotting using tau-specific antibodies. For each of three mutations, abnormal tau with an apparent Mr of 64 and 69 was observed. The dephosphorylated material comigrated with tau isoforms containing exon 10 having four MT-binding repeats but not with 3Rtau. Thus, the brains of patients with both the missense mutations and the splice junction mutation contain aggregates of insoluble 4Rtau in filamentous inclusions, which may lead to neurodegeneration.

Production of identical sextuplet mice by transferring metaphase nuclei from four-cell embryos

Mouse clones were produced by serial nuclear transfer commencing with the transfer of four-cell nuclei at metaphase into unfertilized ooplasts. The donor four-cell-stage nuclei were synchronized in metaphase with nocodazole. The oocytes receiving a four-cell nucleus at metaphase formed two nuclei after artificial activation and inhibition of cytokinesis with cytochalasin B. To obtain embryos with diploid sets of chromosomes, nuclei from each reconstructed embryo were transferred individually into separate enucleated fertilized one-cell embryos, thus doubling the number of identical embryos. This procedure produced a high frequency of development of reconstructed embryos to the blastocyst stage. Of 11 sets of identical embryos produced by serial nuclear transplantation, 83% developed into blastocysts, including three sets of identical septuplet blastocysts. After transfer to recipient mice, a total of 25 (57%) live young were obtained, which included one set of identical sextuplet and two sets of identical quadruplet mice.

Spontaneous mutations recovered as mosaics in the mouse specific-locus test

The specific-locus test (SLT) detects new mutants among mice heterozygous for seven recessive visible markers. Spontaneous mutations can be manifested not only as singleton whole-body mutants in controls (for which we report new data), but as mosaics—either visible (manifesting mottled coat color) in the scored generation (G2) or masked, among the wild-type parental generation (G1). Masked G1 mosaics reveal themselves by producing clusters of whole-body mutants in G2. We provide evidence that most, if not all, mosaics detected in the SLT (both radiation and control progenies) result from a single-strand spontaneous mutation subsequent to the last premeiotic mitosis and before the first postmeiotic one of a parental genome—the “perigametic interval.” Such events in the genomes of the G1 and G0 result, respectively, in visible and masked 50:50 mosaics. Per cell cycle, the spontaneous mutation rate in the perigametic interval is much higher than that in pregamete mitotic divisions. A clearly different locus spectrum further supports the hypothesis of different origin, and casts further doubt on the validity of the doubling-dose risk-estimation method. Because mosaics cannot have arisen in mitotic germ cells, and are not induced by radiation exposure in the perigametic interval, they should not be included in calculations of radiation-induced germ-line mutation rates. For per-generation calculations, inclusion of mosaics yields a spontaneous frequency 1.7 times that calculated from singletons alone for mutations contributed by males; including both sexes, the multiple is 2.2.

Characterization of human cardiac Na+ channel mutations in the congenital long QT syndrome

The congenital long QT syndrome (LQTS) is an inherited disorder characterized by a prolonged cardiac action potential. This delay in cellular repolarization can lead to potentially fatal arrhythmias. One form of LQTS (LQT3) has been linked to the human cardiac voltage-gated sodium channel gene (SCN5A). Three distinct mutations have been identified in the sodium channel gene. The biophysical and functional characteristics of each of these mutant channels were determined by heterologous expression of a recombinant human heart sodium channel in a mammalian cell line. Each mutation caused a sustained, non-inactivating sodium current amounting to a few percent of the peak inward sodium current, observable during long (>50 msec) depolarizations. The voltage dependence and rate of inactivation were altered, and the rate of recovery from inactivation was changed compared with wild-type channels. These mutations in diverse regions of the ion channel protein, all produced a common defect in channel gating that can cause the long QT phenotype. The sustained inward current caused by these mutations will prolong the action potential. Furthermore, they may create conditions that promote arrhythmias due to prolonged depolarization and the altered recovery from inactivation. These results provide insights for successful intervention in the disease.

Candida albicans strains heterozygous and homozygous for mutations in mitogen-activated protein kinase signaling components have defects in hyphal development

The Candida albicans genes, CST20 and HST7, were cloned by their ability to suppress the mating defects of Saccharomyces cerevisiae mutants in the ste20 and ste7 genes, which code for elements of the mating mitogen-activated protein (MAP) kinase pathway. These Candida genes are both structural and functional homologs of the cognate Saccharomyces genes. The pattern of suppression in Saccharomyces is related to their presumptive position in the MAP kinase cascade. Null alleles of these genes were constructed in Candida. The Candida homozygous null mutants are defective in hyphal formation on some media, but are still induced to form hyphae by serum, showing that serum induction of hyphae is independent of the MAP kinase cascade. The Candida heterozygotes CST20/cst20 and HST7/hst7 are also defective in hyphal formation. This lack of dominance of the wild-type allele suggests that gene dosage is important in Candida.

Truncated BRCA2 is cytoplasmic: Implications for cancer-linked mutations

BRCA2 mutations predispose carriers mainly to breast cancer. The vast majority of BRCA2 mutations are predicted to result in a truncated protein product. The smallest known cancer-associated deletion removes from the C terminus only 224 of the 3,418 residues constituting BRCA2, suggesting that these terminal amino acids are crucial for BRCA2 function. A series of green fluorescent protein (GFP)-tagged BRCA2 deletion mutants revealed that nuclear localization depends on two nuclear localization signals that reside within the final 156 residues of BRCA2. Consistent with this observation, an endogenous truncated BRCA2 mutant (6174delT) was found to be cytoplasmic. Together, these studies provide a simple explanation for why the vast majority of BRCA2 mutants are nonfunctional: they do not translocate into the nucleus.

Genghis Khan (Gek) as a putative effector for Drosophila Cdc42 and regulator of actin polymerization

The small GTPases Cdc42 and Rac regulate a variety of biological processes, including actin polymerization, cell proliferation, and JNK/mitogen-activated protein kinase activation, conceivably via distinct effectors. Whereas the effector for mitogen-activated protein kinase activation appears to be p65PAK, the identity of effector(s) for actin polymerization remains unclear. We have found a putative effector for Drosophila Cdc42, Genghis Khan (Gek), which binds to Dcdc42 in a GTP-dependent and effector domain-dependent manner. Gek contains a predicted serine/threonine kinase catalytic domain that is 63% identical to human myotonic dystrophy protein kinase and has protein kinase activities. It also possesses a large coiled-coil domain, a putative phorbol ester binding domain, a pleckstrin homology domain, and a Cdc42 binding consensus sequence that is required for its binding to Dcdc42. To study the in vivo function of gek, we generated mutations in the Drosophila gek locus. Egg chambers homozygous for gek mutations exhibit abnormal accumulation of F-actin and are defective in producing fertilized eggs. These phenotypes can be rescued by a wild-type gek transgene. Our results suggest that this multidomain protein kinase is an effector for the regulation of actin polymerization by Cdc42.

Functional analyses of troponin T mutations that cause hypertrophic cardiomyopathy: Insights into disease pathogenesis and troponin function

Mutations in a number of cardiac sarcomeric protein genes cause hypertrophic cardiomyopathy (HCM). Previous findings indicate that HCM-causing mutations associated with a truncated cardiac troponin T (TnT) and missense mutations in the β-myosin heavy chain share abnormalities in common, acting as dominant negative alleles that impair contractile performance. In contrast, Lin et al. [Lin, D., Bobkova, A., Homsher, E. & Tobacman, L. S. (1996) J. Clin. Invest. 97, 2842–2848] characterized a TnT point mutation (Ile79Asn) and concluded that it might lead to hypercontractility and, thus, potentially a different mechanism for HCM pathogenesis. In this study, three HCM-causing cardiac TnT mutations (Ile79Asn, Arg92Gln, and ΔGlu160) were studied in a myotube expression system. Functional studies of wild-type and mutant transfected myotubes revealed that all three mutants decreased the calcium sensitivity of force production and that the two missense mutations (Ile79Asn and Arg92Gln) increased the unloaded shortening velocity nearly 2-fold. The data demonstrate that TnT can alter the rate of myosin cross-bridge detachment, and thus the troponin complex plays a greater role in modulating muscle contractile performance than was recognized previously. Furthermore, these data suggest that these TnT mutations may cause disease via an increased energetic load on the heart. This would represent a second paradigm for HCM pathogenesis.

Specific mutations in a viral RNA pseudoknot drastically change ribosomal frameshifting efficiency

Many viruses regulate protein synthesis by −1 ribosomal frameshifting using an RNA pseudoknot. Frameshifting is vital for viral reproduction. Using the information gained from the recent high-resolution crystal structure of the beet western yellow virus pseudoknot, a systematic mutational analysis has been carried out in vitro and in vivo. We find that specific nucleotide tertiary interactions at the junction between the two stems of the pseudoknot are crucial. A triplex is found between stem 1 and loop 2, and triplex interactions are required for frameshifting function. For some mutations, loss of one hydrogen bond is sufficient to abolish frameshifting. Furthermore, mutations near the 5′ end of the pseudoknot can increase frameshifting by nearly 300%, possibly by modifying ribosomal contacts. It is likely that the selection of suitable mutations can thus allow viruses to adjust frameshifting efficiencies and thereby regulate protein synthesis in response to environmental change.

Suppression of an absolute defect in Type IV pilus biogenesis by loss-of-function mutations in pilT, a twitching motility gene in Neisseria gonorrhoeae

Type IV pili of Neisseria gonorrhoeae, the Gram-negative etiologic agent of gonorrhea, facilitate colonization of the human host. Gonococcal PilT, a protein belonging to a large family of molecules sharing a highly conserved nucleotide binding domain motif, has been shown to be dispensable for organelle biogenesis but essential for twitching motility and competence for genetic transformation. Here, we show that the defect in pilus biogenesis resulting from mutations in the pilC gene, encoding a putative pilus-associated adhesin for human tissue, can be suppressed by the absence of functional PilT. These data conclusively demonstrate that PilT influences the Type IV pilus biogenesis pathway and strongly suggest that organelle expression is a dynamic process. In addition, these findings imply that PilT antagonizes the process of organelle biogenesis and provide the basis for a model for how the counteractive roles of PilT and PilC might relate mechanistically to the phenomenon of twitching motility.

Scanning mutagenesis of the putative transmembrane segments of Kir2.1, an inward rectifier potassium channel

Structural models of inward rectifier K+ channels incorporate four identical or homologous subunits, each of which has two hydrophobic segments (M1 and M2) which are predicted to span the membrane as α helices. Since hydrophobic interactions between proteins and membrane lipids are thought to be generally of a nonspecific nature, we attempted to identify lipid-contacting residues in Kir2.1 as those which tolerate mutation to tryptophan, which has a large hydrophobic side chain. Tolerated mutations were defined as those which produced measurable inwardly rectifying currents in Xenopus oocytes. To distinguish between water-accessible positions and positions adjacent to membrane lipids or within the protein interior we also mutated residues in M1 and M2 individually to aspartate, since an amino acid with a charged side chain should not be tolerated at lipid-facing or interior positions, due to the energy cost of burying a charge in a hydrophobic environment. Surprisingly, 17 out of 20 and 17 out of 22 non-tryptophan residues in M1 and M2, respectively, tolerated being mutated to tryptophan. Moreover, aspartate was tolerated at 15 out of 22 and 15 out of 21 non-aspartate M1 and M2 positions respectively. Periodicity in the pattern of tolerated vs. nontolerated mutations consistent with α helices or β strands did not emerge convincingly from these data. We consider the possibility that parts of M1 and M2 may be in contact with water.

HSP70 homolog functions in cell-to-cell movement of a plant virus

Plant closteroviruses encode a homolog of the HSP70 (heat shock protein, 70 kDa) family of cellular proteins. To facilitate studies of the function of HSP70 homolog (HSP70h) in viral infection, the beet yellows closterovirus (BYV) was modified to express green fluorescent protein. This tagged virus was competent in cell-to-cell movement, producing multicellular infection foci similar to those formed by the wild-type BYV. Inactivation of the HSP70h gene by replacement of the start codon or by deletion of 493 codons resulted in complete arrest of BYV translocation from cell to cell. Identical movement-deficient phenotypes were observed in BYV variants possessing HSP70h that lacked the computer-predicted ATPase domain or the C-terminal domain, or that harbored point mutations in the putative catalytic site of the ATPase. These results demonstrate that the virus-specific member of the HSP70 family of molecular chaperones functions in intercellular translocation and represents an additional type of a plant viral-movement protein.

The reactive site loop of the serpin SCCA1 is essential for cysteine proteinase inhibition

The high-molecular-weight serine proteinase inhibitors (serpins) are restricted, generally, to inhibiting proteinases of the serine mechanistic class. However, the viral serpin, cytokine response modifier A, and the human serpins, antichymotrypsin and squamous cell carcinoma antigen 1 (SCCA1), inhibit different members of the cysteine proteinase class. Although serpins employ a mobile reactive site loop (RSL) to bait and trap their target serine proteinases, the mechanism by which they inactivate cysteine proteinases is unknown. Our previous studies suggest that SCCA1 inhibits papain-like cysteine proteinases in a manner similar to that observed for serpin–serine proteinase interactions. However, we could not preclude the possibility of an inhibitory mechanism that did not require the serpin RSL. To test this possibility, we employed site-directed mutagenesis to alter the different residues within the RSL. Mutations to either the hinge or the variable region of the RSL abolished inhibitory activity. Moreover, RSL swaps between SCCA1 and the nearly identical serpin, SCCA2 (an inhibitor of chymotrypsin-like serine proteinases), reversed their target specificities. Thus, there were no unique motifs within the framework of SCCA1 that independently accounted for cysteine proteinase inhibitory activity. Collectively, these data suggested that the sequence and mobility of the RSL of SCCA1 are essential for cysteine proteinase inhibition and that serpins are likely to utilize a common RSL-dependent mechanism to inhibit both serine and cysteine proteinases.