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.

Splicing is required for rapid and efficient mRNA export in metazoans

Pre-mRNA splicing is among the last known nuclear events before export of mature mRNA to the cytoplasm. At present, it is not known whether splicing and mRNA export are biochemically coupled processes. In this study, we have injected pre-mRNAs containing a single intron or the same mRNAs lacking an intron (Δi-mRNAs) into Xenopus oocyte nuclei. We find that the spliced mRNAs are exported much more rapidly and efficiently than the identical Δi-mRNAs. Moreover, competition studies using excess Δi-mRNA indicate that different factor(s) are involved in the inefficient export of Δi-mRNA vs. the efficient export of spliced mRNA. Consistent with this conclusion, spliced mRNA and Δi-mRNA, though identical in sequence, are assembled into different messenger ribonucleoprotein particles (mRNP) in vitro. Strikingly, the mRNA in the spliced mRNP, but not in the Δi-mRNP, is exported rapidly and efficiently. We conclude that splicing generates a specific nucleoprotein complex that targets mRNA for export. Our results, revealing a link between splicing and efficient mRNA export, may explain the reports that an intron is required for efficient expression of many protein-coding genes in metazoans.

Alternative splicing, muscle calcium sensitivity, and the modulation of dragonfly flight performance

Calcium sensitivity of myosin cross-bridge activation in striated muscles commonly varies during ontogeny and in response to alterations in muscle usage, but the consequences for whole-organism physiology are not well known. Here we show that the relative abundances of alternatively spliced transcripts of the calcium regulatory protein troponin T (TnT) vary widely in flight muscle of Libellula pulchella dragonflies, and that the mixture of TnT splice variants explains significant portions of the variation in muscle calcium sensitivity, wing-beat frequency, and an index of aerodynamic power output during free flight. Two size-distinguishable morphs differ in their maturational pattern of TnT splicing, yet they show the same relationship between TnT transcript mixture and calcium sensitivity and between calcium sensitivity and aerodynamic power output. This consistency of effect in different developmental and physiological contexts strengthens the hypothesis that TnT isoform variation modulates muscle calcium sensitivity and whole-organism locomotor performance. Modulating muscle power output appears to provide the ecologically important ability to operate at different points along a tradeoff between performance and energetic cost.

TAFII mutations disrupt Dorsal activation in the Drosophila embryo

In this study, we present evidence that the Dorsal activator interacts with limiting amounts of the TFIID complex in the Drosophila embryo. In vitro transcription reactions and protein binding assays implicate the TAFII110 and TAFII60 subunits of the TFIID complex in contributing to Dorsal-mediated activation. Mutations in TAFII110 and TAFII60 result in altered patterns of snail and twist transcription in embryos derived from dl/+ females. These results suggest that TAFIIs contribute to the activation of transcription in vivo and support the hypothesis that subunits of TFIID may serve as targets of enhancer binding proteins.

Inactivating mutations in an SH2 domain-encoding gene in X-linked lymphoproliferative syndrome

X-linked lymphoproliferative syndrome (XLP) is an inherited immunodeficiency characterized by increased susceptibility to Epstein–Barr virus (EBV). In affected males, primary EBV infection leads to the uncontrolled proliferation of virus-containing B cells and reactive cytotoxic T cells, often culminating in the development of high-grade lymphoma. The XLP gene has been mapped to chromosome band Xq25 through linkage analysis and the discovery of patients harboring large constitutional genomic deletions. We describe here the presence of small deletions and intragenic mutations that specifically disrupt a gene named DSHP in 6 of 10 unrelated patients with XLP. This gene encodes a predicted protein of 128 amino acids composing a single SH2 domain with extensive homology to the SH2 domain of SHIP, an inositol polyphosphate 5-phosphatase that functions as a negative regulator of lymphocyte activation. DSHP is expressed in transformed T cell lines and is induced following in vitro activation of peripheral blood T lymphocytes. Expression of DSHP is restricted in vivo to lymphoid tissues, and RNA in situ hybridization demonstrates DSHP expression in activated T and B cell regions of reactive lymph nodes and in both T and B cell neoplasms. These observations confirm the identity of DSHP as the gene responsible for XLP, and suggest a role in the regulation of lymphocyte activation and proliferation. Induction of DSHP may sustain the immune response by interfering with SHIP-mediated inhibition of lymphocyte activation, while its inactivation in XLP patients results in a selective immunodeficiency to EBV.

Control of alternative pre-mRNA splicing by distributed pentameric repeats

Multiple copies of the hexamer TGCATG have been shown to regulate fibronectin pre-mRNA alternative splicing. GCATG repeats also are clustered near the regulated calcitonin-specific 3′ splice site in the rat calcitonin/CGRP gene. Specific mutagenesis of these repeats in calcitonin/CGRP pre-mRNA resulted in the loss of calcitonin-specific splicing, suggesting that the native repeats act to enhance alternative exon inclusion. Mutation of subsets of these elements implies that alternative splicing requires a minimum of two repeats, and that the combination of one intronic and one exonic repeat is necessary for optimal cell-specific splicing. However, multimerized intronic repeats inhibited calcitonin-specific splicing in both the wild-type context and in a transcript lacking endogenous repeats. These results suggest that both the number and distribution of repeats may be important features for the regulation of tissue-specific alternative splicing. Further, RNA containing a single repeat bound cell-specific protein complexes, but tissue-specific differences in protein binding were not detected by using multimerized repeats. Together, these data support a novel model for alternative splicing regulation that requires the cell-specific recognition of multiple, distributed sequence elements.

Hereditary hemochromatosis: Effects of C282Y and H63D mutations on association with β2-microglobulin, intracellular processing, and cell surface expression of the HFE protein in COS-7 cells

Hereditary hemochromatosis (HH) is the most common autosomal recessive disorder known in humans. A candidate gene for HH called HFE has recently been cloned that encodes a novel member of the major histocompatibility complex class I family. Most HH patients are homozygous for a Cys-282→Tyr (C282Y) mutation in HFE gene, which has been shown to disrupt interaction with β2-microglobulin; a second mutation, His-63→Asp (H63D), is enriched in HH patients who are heterozygous for C282Y mutation. The aims of this study were to determine the effects of the C282Y and H63D mutations on the cellular trafficking and degradation of the HFE protein in transfected COS-7 cells. The results indicate that, while the wild-type and H63D HFE proteins associate with β2-microglobulin and are expressed on the cell surface of COS-7 cells, these capabilities are lost by the C282Y HFE protein. We present biochemical and immunofluorescence data that indicate that the C282Y mutant protein: (i) is retained in the endoplasmic reticulum and middle Golgi compartment, (ii) fails to undergo late Golgi processing, and (iii) is subject to accelerated degradation. The block in intracellular transport, accelerated turnover, and failure of the C282Y protein to be presented normally on the cell surface provide a possible basis for impaired function of this mutant protein in HH.

Double muscling in cattle due to mutations in the myostatin gene

Myostatin (GDF-8) is a member of the transforming growth factor β superfamily of secreted growth and differentiation factors that is essential for proper regulation of skeletal muscle mass in mice. Here we report the myostatin sequences of nine other vertebrate species and the identification of mutations in the coding sequence of bovine myostatin in two breeds of double-muscled cattle, Belgian Blue and Piedmontese, which are known to have an increase in muscle mass relative to conventional cattle. The Belgian Blue myostatin sequence contains an 11-nucleotide deletion in the third exon which causes a frameshift that eliminates virtually all of the mature, active region of the molecule. The Piedmontese myostatin sequence contains a missense mutation in exon 3, resulting in a substitution of tyrosine for an invariant cysteine in the mature region of the protein. The similarity in phenotypes of double-muscled cattle and myostatin null mice suggests that myostatin performs the same biological function in these two species and is a potentially useful target for genetic manipulation in other farm animals.

Intron self-complementarity enforces exon inclusion in a yeast pre-mRNA

Skipping of internal exons during removal of introns from pre-mRNA must be avoided for proper expression of most eukaryotic genes. Despite significant understanding of the mechanics of intron removal, mechanisms that ensure inclusion of internal exons in multi-intron pre-mRNAs remain mysterious. Using a natural two-intron yeast gene, we have identified distinct RNA–RNA complementarities within each intron that prevent exon skipping and ensure inclusion of internal exons. We show that these complementarities are positioned to act as intron identity elements, bringing together only the appropriate 5′ splice sites and branchpoints. Destroying either intron self-complementarity allows exon skipping to occur, and restoring the complementarity using compensatory mutations rescues exon inclusion, indicating that the elements act through formation of RNA secondary structure. Introducing new pairing potential between regions near the 5′ splice site of intron 1 and the branchpoint of intron 2 dramatically enhances exon skipping. Similar elements identified in single intron yeast genes contribute to splicing efficiency. Our results illustrate how intron secondary structure serves to coordinate splice site pairing and enforce exon inclusion. We suggest that similar elements in vertebrate genes could assist in the splicing of very large introns and in the evolution of alternative splicing.

Conserved biological function between Pax-2 and Pax-5 in midbrain and cerebellum development: Evidence from targeted mutations

The development of two major subdivisions of the vertebrate nervous system, the midbrain and the cerebellum, is controlled by signals emanating from a constriction in the neural primordium called the midbrain/hindbrain organizer (Joyner, A. L. (1996) Trends Genet. 12, 15–201). The closely related transcription factors Pax-2 and Pax-5 exhibit an overlapping expression pattern very early in the developing midbrain/hindbrain junction. Experiments carried out in fish (Krauss, S., Maden, M., Holder, N. & Wilson, S. W. (1992) Nature (London) 360, 87–89) with neutralizing antibodies against Pax-b, the orthologue of Pax-2 in mouse, placed this gene family in an regulatory cascade necessary for the development of the midbrain and the cerebellum. The targeted mutation of Pax-5 has been reported to have only slight effects in the development of structures derived from the isthmic constriction, whereas the Pax-2 null mutant mice show a background-dependent phenotype with varying penetrance. To test a possible redundant function between Pax-2 and Pax-5 we analyzed the brain phenotypes of mice expressing different dosages of both genes. Our results demonstrate a conserved biological function of both proteins in midbrain/hindbrain regionalization. Additionally, we show that one allele of Pax-2, but not Pax-5, is necessary and sufficient for midbrain and cerebellum development in C57BL/6 mice.

A genetic screen for mutations that disrupt an auditory response in Drosophila melanogaster

Hearing is one of the last sensory modalities to be subjected to genetic analysis in Drosophila melanogaster. We describe a behavioral assay for auditory function involving courtship among groups of males triggered by the pulse component of the courtship song. In a mutagenesis screen for mutations that disrupt the auditory response, we have recovered 15 mutations that either reduce or abolish this response. Mutant audiograms indicate that seven mutants reduced the amplitude of the response at all intensities. Another seven abolished the response altogether. The other mutant, 5L3, responded only at high sound intensities, indicating that the threshold was shifted in this mutant. Six mutants were characterized in greater detail. 5L3 had a general courtship defect; courtship of females by 5L3 males also was affected strongly. 5P1 males courted females normally but had reduced success at copulation. 5P1 and 5N18 showed a significant decrement in olfactory response, indicating that the defects in these mutations are not specific to the auditory pathway. Two other mutants, 5M8 and 5N30, produced amotile sperm although in 5N30 this phenotype was genetically separable from the auditory phenotype. Finally, a new adult circling behavior phenotype, the pirouette phenotype, associated with massive neurodegeneration in the brain, was discovered in two mutants, 5G10 and 5N18. This study provides the basis for a genetic and molecular dissection of auditory mechanosensation and auditory behavior.

Hair cell-specific splicing of mRNA for the α1D subunit of voltage-gated Ca2+ channels in the chicken’s cochlea

The L-type voltage-gated Ca2+ channels that control tonic release of neurotransmitter from hair cells exhibit unusual electrophysiological properties: a low activation threshold, rapid activation and deactivation, and a lack of Ca2+-dependent inactivation. We have inquired whether these characteristics result from cell-specific splicing of the mRNA for the L-type α1D subunit that predominates in hair cells of the chicken’s cochlea. The α1D subunit in hair cells contains three uncommon exons: one encoding a 26-aa insert in the cytoplasmic loop between repeats I and II, an alternative exon for transmembrane segment IIIS2, and a heretofore undescribed exon specifying a 10-aa insert in the cytoplasmic loop between segments IVS2 and IVS3. We propose that the alternative splicing of the α1D mRNA contributes to the unusual behavior of the hair cell’s voltage-gated Ca2+ channels.

Ancient mtDNA sequences in the human nuclear genome: A potential source of errors in identifying pathogenic mutations

Nuclear-localized mtDNA pseudogenes might explain a recent report describing a heteroplasmic mtDNA molecule containing five linked missense mutations dispersed over the contiguous mtDNA CO1 and CO2 genes in Alzheimer’s disease (AD) patients. To test this hypothesis, we have used the PCR primers utilized in the original report to amplify CO1 and CO2 sequences from two independent ρ° (mtDNA-less) cell lines. CO1 and CO2 sequences amplified from both of the ρ° cells, demonstrating that these sequences are also present in the human nuclear DNA. The nuclear pseudogene CO1 and CO2 sequences were then tested for each of the five “AD” missense mutations by restriction endonuclease site variant assays. All five mutations were found in the nuclear CO1 and CO2 PCR products from ρ° cells, but none were found in the PCR products obtained from cells with normal mtDNA. Moreover, when the overlapping nuclear CO1 and CO2 PCR products were cloned and sequenced, all five missense mutations were found, as well as a linked synonymous mutation. Unlike the findings in the original report, an additional 32 base substitutions were found, including two in adjacent tRNAs and a two base pair deletion in the CO2 gene. Phylogenetic analysis of the nuclear CO1 and CO2 sequences revealed that they diverged from modern human mtDNAs early in hominid evolution about 770,000 years before present. These data would be consistent with the interpretation that the missense mutations proposed to cause AD may be the product of ancient mtDNA variants preserved as nuclear pseudogenes.

Ultrastructural Analysis of Transcription and Splicing in the Cell Nucleus after Bromo-UTP Microinjection

In this study we demonstrate, at an ultrastructural level, the in situ distribution of heterogeneous nuclear RNA transcription sites after microinjection of 5-bromo-UTP (BrUTP) into the cytoplasm of living cells and subsequent postembedding immunoelectron microscopic visualization after different labeling periods. Moreover, immunocytochemical localization of several pre-mRNA transcription and processing factors has been carried out in the same cells. This high-resolution approach allowed us to reveal perichromatin regions as the most important sites of nucleoplasmic RNA transcription and the perichromatin fibrils (PFs) as in situ forms of nascent transcripts. Furthermore, we show that transcription takes place in a rather diffuse pattern, without notable local accumulation of transcription sites. RNA polymerase II, heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins, general transcription factor TFIIH, poly(A) polymerase, splicing factor SC-35, and Sm complex of small nuclear ribonucleoproteins (snRNPs) are associated with PFs. This strongly supports the idea that PFs are also sites of major pre-mRNA processing events. The absence of nascent transcripts, RNA polymerase II, poly(A) polymerase, and hnRNPs within the clusters of interchromatin granules rules out the possibility that this domain plays a role in pre-mRNA transcription and polyadenylation; however, interchromatin granule-associated zones contain RNA polymerase II, TFIIH, and Sm complex of snRNPs and, after longer periods of BrUTP incubation, also Br-labeled RNA. Their role in nuclear functions still remains enigmatic. In the nucleolus, transcription sites occur in the dense fibrillar component. Our fine structural results show that PFs represent the major nucleoplasmic structural domain involved in active pre-mRNA transcriptional and processing events.

Mutations at Phosphorylation Sites of Xenopus Microtubule-associated Protein 4 Affect Its Microtubule-binding Ability and Chromosome Movement during Mitosis

Microtubule-associated proteins (MAPs) bind to and stabilize microtubules (MTs) both in vitro and in vivo and are thought to regulate MT dynamics during the cell cycle. It is known that p220, a major MAP of Xenopus, is phosphorylated by p34cdc2 kinase as well as MAP kinase in mitotic cells, and that the phosphorylated p220 loses its MT-binding and -stabilizing abilities in vitro. We cloned a full-length cDNA encoding p220, which identified p220 as a Xenopus homologue of MAP4 (XMAP4). To examine the physiological relevance of XMAP4 phosphorylation in vivo, Xenopus A6 cells were transfected with cDNAs encoding wild-type or various XMAP4 mutants fused with a green fluorescent protein. Mutations of serine and threonine residues at p34cdc2 kinase-specific phosphorylation sites to alanine interfered with mitosis-associated reduction in MT affinity of XMAP4, and their overexpression affected chromosome movement during anaphase A. These findings indicated that phosphorylation of XMAP4 (probably by p34cdc2 kinase) is responsible for the decrease in its MT-binding and -stabilizing abilities during mitosis, which are important for chromosome movement during anaphase A.