1. The M2 protein of influenza A virus is implicated in transmembrane pH regulation during infection. Whole-cell patch clamp of mouse erythroleukaemia cells expressing the M2 protein in the surface membrane showed a conductance due to M2 which was specifically blocked by the anti-influenza drug rimantadine. 2. The ion selectivity of the rimantadine-sensitive current through M2 was determined.Reversal potentials were close to equilibrium potentials for transmembrane pH gradients and not to those for Na+, K+ or Cl-concentration gradients. M2 permeability to Na+ relative to H+ was estimated to be less than 6 x 10-7.3. The M2 conductance increased as external pH decreased below 8X5 and approached saturation at an external pH of 4, effects attributable to increased permeability due to increased driving potential and to activation by low external pH. Both activation and permeation could be described by interaction of protons with sites on M2, with apparent dissociation constants of approximately 0.1 /SM and 1 /SM, respectively, under physiological conditions.4. The M2 protein can transfer protons selectively across membranes with the H+ electrochemical gradient, properties consistent with its role in modifying virion and trans-
We describe here for the first time the site of retention within the nucleus of pre-mRNA processing mutants unable to be exported to the cytoplasm. Fluorescence in situ hybridization was used to detect transcripts from human β-globin genes that are either normal or defective in splicing or 3Ј end formation. Nuclear transcripts of both wild-type and mutant RNAs are detected only as intranuclear foci that colocalize with the template gene locus. The kinetics of transcript release from the site of transcription was assessed by treatment of cells with the transcriptional inhibitors actinomycin D, α-amanitin and DRB. These drugs induce the rapid disappearance of nuclear foci corresponding to wild-type human β-globin RNA. In contrast, pre-mRNA mutants defective in either splicing or 3Ј end formation and which fail to be transported to the cytoplasm, are retained at the site of transcription. Therefore, 3Ј end processing and splicing appear to be rate limiting for release of mRNA from the site of transcription.
The M2 protein of influenza A viruses forms a proton channel involved in modifying virion and trans Golgi pH during infection. Previous studies of the proton current using whole‐cell patch clamp of mouse erythroleukaemia (MEL) cells expressing the M2 protein of the ‘Weybridge’ strain provided evidence for two protonation sites, one involved in permeation, the other in activation by acid pH. The present report compares the M2 channels of two different strains of influenza virus, ‘Weybridge’ (WM2) and ‘Rostock’ (RM2). Whereas with external acid pH the current‐voltage relations showed similar small degrees of inward rectification, a similar apparent Kd of approximately 10 μm for proton permeation and a high selectivity for protons over Na+, the two M2 proteins differed in whole‐cell conductance at low and high pH. The proton conductance of unit membrane area was on average 7‐fold greater in RM2‐ than WM2‐expressing MEL cells. At high external pH WM2 was shown previously to have small conductance for outward current at positive driving potential. In contrast, RM2 shows high conductance for outward current with high external pH, but shows small conductance for inward current with high internal pH, conditions in which WM2 shows high conductance for inward current. The different properties of the conductances due to the two channels at high pH were determined by three amino acids in their transmembrane domains. All intermediate mutants possessed one or other property and transformation of the WM2 phenotype into that of RM2 required substitution in all three residues V27I, F38L and D44N; single substitutions in RM2 effected the opposite phenotypic change. The significance of this difference for virus replication is not clear and it may be that the higher proton flux associated with RM2 is the main factor determining its increased ability to dissipate pH gradients. It is apparent, however, from the specific differences in the sidedness of the pH‐induced changes in voltage dependence of the whole‐cell current that this is an intrinsic property of the virus proton channel which may have parallels with regulation of other proton channels.
The second intron (betaIVS-II) of the human beta-globin gene is essential for the accumulation of stable cytoplasmic mRNA and is implicated in promoting efficient 3'-end formation. This report presents quantitative comparisons between betaIVS-II mutants at physiological levels of expression from within a natural chromatin context in vivo which further defines it's function. In marked contrast to a beta-globin gene lacking a second intron, two mutants defective in splicing (small size or a splice donor mutation), still undergo essentially normal levels of 3'-end formation and in the absence of exon skipping. Therefore, 3' cleavage of beta-globin transcripts requires the presence of betaIVS-II sequences, but not the splicing reaction. The placement of betaIVS-II in the IVS-I position did not reduce the efficiency of 3' cleavage indicating that the distance between the necessary element(s) in this intron and the polyadenylation recognition site is not a crucial factor. Subsequent placement of betaIVS-I in the intron II position, reduced the efficiency of 3'-end formation to only 16% of normal. A direct replacement of intron II by the heterologous introns betaIVS-I or alpha-globin IVS-II, only partially substitute (16 and 30% respectively) for betaIVS-II. Hybrid introns show that efficient 3'-end formation is strongly enhanced by the presence of the terminal 60 nt of betaIVS-II. These data imply that the last intervening sequence of multiple intron containing genes is a principal determinant of the efficiency of 3'-end formation and may act as a post-transcriptional regulatory step in gene expression.
Splicing and 3′-end processing (including cleavage and polyadenylation) of vertebrate pre-mRNAs are tightly coupled events that contribute to the extensive molecular network that coordinates gene expression. Sequences within the terminal intron of genes are essential to stimulate pre-mRNA 3′-end processing, although the factors mediating this effect are unknown. Here, we show that the pyrimidine tract of the last splice acceptor site of the human β-globin gene is necessary to stimulate mRNA 3′-end formation in vivo and binds the U2AF 65 splicing factor. Naturally occurring β-thalassaemia-causing mutations within the pyrimidine tract reduces both U2AF 65 binding and 3′-end cleavage efficiency. Significantly, a fusion protein containing U2AF 65, when tethered upstream of a cleavage/polyadenylation site, increases 3′-end cleavage efficiency in vitro and in vivo. Therefore, we propose that U2AF 65 promotes 3′-end processing, which contributes to 3′-terminal exon definition.
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