A suppressor of a yeast splicing mutation (prp8-1) encodes a putative ATP-dependent RNA helicase

Five small nuclear RNAs (snRNAs) are required for nuclear pre-messenger RNA splicing: U1, U2, U4, U5 and U6. The yeast U1 and U2 snRNAs base-pair to the 5' splice site and branch-point sequences of introns respectively. The role of the U5 and U4/U6 small nuclear ribonucleoprotein particles (snRNPs) in splicing is not clear, though a catalytic role for the U6 snRNA has been proposed. Less is known about yeast splicing factors, but the availability of genetic techniques in Saccharomyces cerevisiae has led to the identification of mutants deficient in nuclear pre-mRNA splicing (prp2-prp27). Several PRP genes have now been cloned and their protein products characterized. The PRP8 protein is a component of the U5 snRNP and associates with the U4/U6 snRNAs/snRNP to form a multi-snRNP particle believed to be important for spliceosome assembly. We have isolated extragenic suppressors of the prp8-1 mutation of S. cerevisiae and present here the preliminary characterization of one of these suppressors, spp81. The predicted amino-acid sequence of the SPP81 protein shows extensive similarity to a recently identified family of proteins thought to possess ATP-dependent RNA helicase activity. The possible role of this putative helicase in nuclear pre-mRNA splicing is discussed.     

BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain

Neuropathic pain that occurs after peripheral nerve injury depends on the hyperexcitability of neurons in the dorsal horn of the spinal cord. Spinal microglia stimulated by ATP contribute to tactile allodynia, a highly debilitating symptom of pain induced by nerve injury. Signalling between microglia and neurons is therefore an essential link in neuropathic pain transmission, but how this signalling occurs is unknown. Here we show that ATP-stimulated microglia cause a depolarizing shift in the anion reversal potential (E(anion)) in spinal lamina I neurons. This shift inverts the polarity of currents activated by GABA (gamma-amino butyric acid), as has been shown to occur after peripheral nerve injury. Applying brain-derived neurotrophic factor (BDNF) mimics the alteration in E(anion). Blocking signalling between BDNF and the receptor TrkB reverses the allodynia and the E(anion) shift that follows both nerve injury and administration of ATP-stimulated microglia. ATP stimulation evokes the release of BDNF from microglia. Preventing BDNF release from microglia by pretreating them with interfering RNA directed against BDNF before ATP stimulation also inhibits the effects of these cells on the withdrawal threshold and E(anion). Our results show that ATP-stimulated microglia signal to lamina I neurons, causing a collapse of their transmembrane anion gradient, and that BDNF is a crucial signalling molecule between microglia and neurons. Blocking this microglia-neuron signalling pathway may represent a therapeutic strategy for treating neuropathic pain.     

Mutations in dynamin 2 cause dominant centronuclear myopathy

Autosomal dominant centronuclear myopathy is a rare congenital myopathy characterized by delayed motor milestones and muscular weakness. In 11 families affected by centronuclear myopathy, we identified recurrent and de novo missense mutations in the gene dynamin 2 (DNM2, 19p13.2), which encodes a protein involved in endocytosis and membrane trafficking, actin assembly and centrosome cohesion. The transfected mutants showed reduced labeling in the centrosome, suggesting that DNM2 mutations might cause centronuclear myopathy by interfering with centrosome function.     

Mutations in ACTN4, encoding alpha-actinin-4, cause familial focal segmental glomerulosclerosis

Focal and segmental glomerulosclerosis (FSGS) is a common, non-specific renal lesion. Although it is often secondary to other disorders, including HIV infection, obesity, hypertension and diabetes, FSGS also appears as an isolated, idiopathic condition. FSGS is characterized by increased urinary protein excretion and decreasing kidney function. Often, renal insufficiency in affected patients progresses to end-stage renal failure, a highly morbid state requiring either dialysis therapy or kidney transplantation. Here we present evidence implicating mutations in the gene encoding alpha-actinin-4 (ACTN4; ref. 2), an actin-filament crosslinking protein, as the cause of disease in three families with an autosomal dominant form of FSGS. In vitro, mutant alpha-actinin-4 binds filamentous actin (F-actin) more strongly than does wild-type alpha-actinin-4. Regulation of the actin cytoskeleton of glomerular podocytes may be altered in this group of patients. Our results have implications for understanding the role of the cytoskeleton in the pathophysiology of kidney disease and may lead to a better understanding of the genetic basis of susceptibility to kidney damage.     

Yeast Sm-like proteins function in mRNA decapping and decay

One of the main mechanisms of messenger RNA degradation in eukaryotes occurs by deadenylation-dependent decapping which leads to 5'-to-3' decay. A family of Sm-like (Lsm) proteins has been identified, members of which contain the 'Sm' sequence motif, form a complex with U6 small nuclear RNA and are required for pre-mRNA splicing. Here we show that mutations in seven yeast Lsm proteins (Lsm1-Lsm7) also lead to inhibition of mRNA decapping. In addition, the Lsm1-Lsm7 proteins co-immunoprecipitate with the mRNA decapping enzyme (Dcp1), a decapping activator (Pat1/Mrt1) and with mRNA. This indicates that the Lsm proteins may promote decapping by interactions with the mRNA and the decapping machinery. In addition, the Lsm complex that functions in mRNA decay appears to be distinct from the U6-associated Lsm complex, indicating that Lsm proteins form specific complexes that affect different aspects of mRNA metabolism.     

Conservation between yeast and man of a protein associated with U5 small nuclear ribonucleoprotein

The process of nuclear pre-messenger RNA splicing is similar in Saccharomyces cerevisiae and metazoan cells in that the two-step mechanism is identical and the reaction occurs in a large ribonucleoprotein complex, the spliceosome. Little is known, however, about the degree of conservation of splicing factors other than of the small nuclear RNAs (snRNAs). Yeast counterparts of the metazoan spliceosomal snRNAs (U1, U2, U4, U5 and U6) have been identified but, with the exception of U6, the yeast snRNAs are larger and sequence similarity is limited to short regions. By using antibodies against the yeast PRP8 protein, a pre-mRNA splicing factor of relative molecular mass 280,000 (Mr280K) stably associated with U5 small nuclear ribonucleoproteins (snRNPs), we have now identified an immunologically related protein in HeLa cell nuclear extracts. The HeLa cell protein has an Mr greater than 200K and is associated with purified 20S U5 snRNPs. This is the first report of phylogenetic conservation between yeast and man of a protein splicing factor.     

Transformation of yeast by a replicating hybrid plasmid.

Chimaeric plasmids have been constructed containing a yeast plasmid and fragments of yeast nuclear DNA linked to pMB9, a derivative of the ColEl plasmid from E. coli. Two plasmids were isolated which complement leuB mutations in E. coli. These plasmids have been used to develop a method for transforming a leu2 strain of S. cerevisiae to Leu+ with high frequency. The yeast transformants contained multiple plasmid copies which were recovered by transformation in E. coli. The yeast plasmid sequence recombined intramolecularly during propagation in yeast.     

Structural diversity and African origin of the 17q21.31 inversion polymorphism

The 17q21.31 inversion polymorphism exists either as direct (H1) or inverted (H2) haplotypes with differential predispositions to disease and selection. We investigated its genetic diversity in 2,700 individuals, with an emphasis on African populations. We characterize eight structural haplotypes due to complex rearrangements that vary in size from 1.08-1.49 Mb and provide evidence for a 30-kb H1-H2 double recombination event. We show that recurrent partial duplications of the KANSL1 gene have occurred on both the H1 and H2 haplotypes and have risen to high frequency in European populations. We identify a likely ancestral H2 haplotype (H2') lacking these duplications that is enriched among African hunter-gatherer groups yet essentially absent from West African populations. Whereas H1 and H2 segmental duplications arose independently and before human migration out of Africa, they have reached high frequencies recently among Europeans, either because of extraordinary genetic drift or selective sweeps.