Human U4/U6.U5 and U4atac/U6atac.U5 tri-snRNPs exhibit similar protein compositions

In the U12-dependent spliceosome, the U4atac/U6atac snRNP represents the functional analogue of the major U4/U6 snRNP. Little information is available presently regarding the protein composition of the former snRNP and its association with other snRNPs. In this report we show that human U4atac/U6atac di-snRNPs associate with U5 snRNPs to form a 25S U4atac/U6atac.U5 trimeric particle. Comparative analysis of minor and major tri-snRNPs by using immunoprecipitation experiments revealed that their protein compositions are very similar, if not identical. Not only U5-specific proteins but, surprisingly, all tested U4/U6- and major tri-snRNP-specific proteins were detected in the minor tri-snRNP complex. Significantly, the major tri-snRNP-specific proteins 65K and 110K, which are required for integration of the major tri-snRNP into the U2-dependent spliceosome, were among those proteins detected in the minor tri-snRNP, raising an interesting question as to how the specificity of addition of tri-snRNP to the corresponding spliceosome is maintained. Moreover, immunodepletion studies demonstrated that the U4/U6-specific 61K protein, which is involved in the formation of major tri-snRNPs, is essential for the association of the U4atac/U6atac di-snRNP with U5 to form the U4atac/U6atac.U5 tri-snRNP. Subsequent immunoprecipitation studies demonstrated that those proteins detected in the minor tri-snRNP complex are also incorporated into U12-dependent spliceosomes. This remarkable conservation of polypeptides between minor and major spliceosomes, coupled with the absence of significant sequence similarity between the functionally analogous snRNAs, supports an evolutionary model in which most major and minor spliceosomal proteins, but not snRNAs, are derived from a common ancestor.     

SR proteins escort the U4/U6.U5 tri-snRNP to the spliceosome.

Pre-spliceosomes, formed in HeLa nuclear extracts and isolated by sedimentation on glycerol gradients, were chased into spliceosomes, the macromolecular enzyme that catalyzes intron removal. We demonstrate that the pre-spliceosome to spliceosome transition was dependent on ATP hydrolysis and required both a U-rich small nuclear ribonucleoprotein (U snRNP)-containing fraction and a fraction of non-snRNP factors. The active components in the non-snRNP fraction were identified as SR proteins and were purified to apparent homogeneity. Recombinant SR proteins (ASF, SC35, SRp55), as well as gel-purified SR proteins, with the exception of SRp20, were able to restore efficient spliceosome formation. We also demonstrate that the pre-spliceosome to spliceosome transition requires phosphorylated SR proteins. This is the first evidence that SR proteins are required for the pre-spliceosome to spliceosome transition, the step at which the U4/U6.U5 tri-snRNP assembles on the pre-mRNA. The results shown here, together with previous data, suggest U snRNPs require SR proteins as escorts to enter the assembling spliceosome.     

Alkaloids of Uncaria attenuata,U. orientalis and U. Canescens

3-Iso-19-epi-ajmalicine, epiallo-corynantheine and dihydrocorynantheine pseudoindoxyl, not previously known as natural products, have been isolated from samples of U. attenuata. Akuammigine, dihydrocorynantheine, hirsutine, hirsuteine, mitraphylline, speciophylline, uncarines A and B, isorhynchophylline rhynchophylline, isocorynoxeine, corynoxeine, corynoxine B, rotundifoline, speciofoline, two yohimbine isomers, a yohimbine oxindole and an unidentified indole alkaloid (M⁺, m/e 347) have been obtained from samples of the same species. 3-Iso-ajmalicine, harmane, isopteropodine, pteropodine, uncarine F, speciophylline, isomitraphylline, mitraphylline and N-oxides of these six oxindole alkaloids have been isolated from samples of U. orientalis. Several samples of U. canescens have yielded harmane while one sample contained the four pteropodine isomers. The variation in the alkaloid content of these three species is discussed.     

Stabilities of consecutive A.C, C.C, G.G, U.C, and U.U mismatches in RNA internal loops: Evidence for stable hydrogen-bonded U.U and C.C.+ pairs.

The stability and structure of RNA duplexes with consecutive A.C, C.A, C.C, G.G, U.C, C.U, and U.U mismatches were studied by UV melting, CD, and NMR. The results are compared to previous results for GA and AA internal loops [SantaLucia, J., Kierzek, R., & Turner, D. H. (1990) Biochemistry 29, 8813-8819; Peritz, A., Kierzek, R., & Turner, D.H. (1991) Biochemistry 30, 6428-6436)]. The observed order for stability increments of internal loop formation at pH 7 is AG = GA approximately UU greater than GG greater than or equal to CA greater than or equal to AA = CU = UC greater than or equal to CC greater than or equal to AC. The results suggest two classes for internal loops with consecutive mismatches: (1) loops that stabilize duplexes and have strong hydrogen bonding and (2) loops that destabilize duplexes and may not have strong hydrogen bonding. Surprisingly, rCGCUUGCG forms a very stable duplex at pH 7 in 1 M NaCl with a TM of 44.8 degrees C at 1 x 10(-4) M and a delta G degrees 37 of -7.2 kcal/mol. NOE studies of the imino protons indicate hydrogen bonding within the U.U mismatches in a wobble-type structure. Resonances corresponding to the hydrogen-bonded uridines are located at 11.3 and 10.4 ppm. At neutral pH, rCGCCCGCG is one of the least stable duplexes with a TM of 33.2 degrees C and delta G degrees 37 of -5.1 kcal/mol. Upon lowering the pH to 5.5, however, the TM increases by 12 degrees C, and delta G degrees 37 becomes more favorable by 2.5 kcal/mol. The pH dependence of rCGCCCGCG may be due to protonation of the internal loop C's, since no changes in thermodynamic parameters are observed for rCGCUUGCG between pH 7 and 5.5. Furthermore, two broad imino proton resonances are observed at 10.85 and 10.05 ppm for rCGCCCGCG at pH 5.3, but not at pH 6.5. This is also consistent with C.C+ base pairs forming at pH 5.5. rCGCCAGCG and rGGCACGCC have a small pH dependence, with TM increases of 5 and 3 degrees C, respectively, upon lowering the pH from 7 to 5.5. rCGCCUGCG and rCGCUCGCG also show little pH dependence, with TM increases of 0.8 and 1.4 degrees C, respectively, upon lowering the pH to 5.5.(ABSTRACT TRUNCATED AT 400 WORDS)     

The yeast Prp3 protein is a U4/U6 snRNP protein necessary for integrity of the U4/U6 snRNP and the U4/U6.U5 tri-snRNP.

Previously, yeast prp3 mutants were found to be blocked prior to the first catalytic step of pre-mRNA splicing. No splicing intermediates or products are formed from pre-mRNA in heat-inactivated prp3 mutants or prp3 mutant extracts. Here we show that Prp3p is a component of the U4/U6 snRNP and is also present in the U4/U6.U5 tri-snRNP. Heat inactivation of prp3 extracts results in depletion of free U6 snRNPs and U4/U6.U5 tri-snRNPs, but not U4/U6 snRNPs or U5 snRNPs. Free U4 snRNP, normally not present in wild-type extracts, accumulates under these conditions. Assays of in vivo levels of snRNAs in a prp3 mutant revealed that amounts of free U6 snRNA decreased, free U4 snRNA increased, and U4/U6 hybrids decreased slightly. These results suggest that Prp3p is required for formation of stable U4/U6 snRNPs and for assembly of the U4/U6.U5 tri-snRNP from its component snRNPs. Upon inactivation of Prp3p, spliceosomes cannot assemble from prespliceosomes due to the absence of intact U4/U6.U5 tri-snRNPs. Prp3p is homologous to a human protein that is a component of U4/U6 snRNPs, exemplifying the conservation of splicing factors between yeast and metazoans.     

Pre-mRNA splicing by complementation with purified human U1, U2, U4/U6 and U5 snRNPs.

The four major nucleoplasmic small nuclear ribonucleoprotein particles U1, U2, U4/U6 and U5 can be extensively purified from HeLa cells by immunoaffinity chromatography using a monoclonal anti-trimethylguanosine antibody. The snRNP particles in active splicing extracts are selectively bound to the immunoaffinity matrix, and are then gently eluted by competition with an excess of free nucleoside. Biochemical complementation studies show that the purified snRNPs are active in pre-mRNA splicing, but only in the presence of additional non-snRNP protein factors. All the RNPs that are necessary for splicing can be purified in this manner. The active snRNPs are characterized with respect to their polypeptide composition, and shown to be distinct from several other activities implicated in splicing.     

U2 RNA shares a structural domain with U1, U4, and U5 RNAs.

We previously reported common structural features within the 3'-terminal regions of U1, U4, and U5 RNAs. To check whether these features also exist in U2 RNA, the primary and secondary structures of the 3'-terminal regions of chicken, pheasant, and rat U2 RNAs were examined. Whereas no difference was observed between pheasant and chicken, the chicken and rat sequences were only 82.5% homologous. Such divergence allowed us to propose a unique model of secondary structure based on maximum base-pairing and secondary structure conservation. The same model was obtained from the results of limited digestion of U2 RNA with various nucleases. Comparison of this structure with those of U1, U4, and U5 RNAs shows that the four RNAs share a common structure designated as domain A, and consisting of a free single-stranded region with the sequence Pu-A-(U)n-G-Pup flanked by two hairpins. The hairpin on the 3' side is very stable and has the sequence Py-N-Py-Gp in the loop. The presence of this common domain is discussed in connection with relationships among U RNAs and common protein binding sites.     

U.K. deep diving trials

Using a breathing medium of 40 kPa oxygen, remainder helium, 18 volunteer subjects participated in a series of 15 exposures to pressures equivalent to depths of 180-540 m s.w. The time of exposure at these pressures was mostly 2 days, except for the 540 m s.w. exposure, when 6 days were spent at full pressure. Compression procedures, based upon placing 'stages' at 60 m s.w. intervals, evolved with experience and proved to be a highly successful way of achieving acceptable pressure-time courses. Decompression combined slow linear release of pressure with overnight halts for sleep. On one occasion a depth of 660 m s.w. was reached by breathing 40 kPa oxygen, 10% nitrogen, remainder helium. Throughout all exposures, teams of investigators followed the changes in cardiovascular, respiratory, haematological, neurophysiological and metabolic status, and mental performance of the volunteers. Some major findings were that the neurophysiological and behavioural changes could be assigned to the motor, or vestibular, or cerebral, or autonomic systems, and were mainly first observed during compression. The subjects suffered, apparently from severe nitrogen narcosis, when breathing 10% (by volume) nitrogen in oxygen-helium at 420 m s.w. Lung ventilation was remarkably adaptable to the oxygen requirements of exercise at all depths, but cardiac output was adversely affected at 540 m s.w., particularly for heavier workloads. Ventilatory responses to carbon dioxide were significantly elevated after diving. Thermal balance was seen to be precarious, but nevertheless it was achieved by the normal subjective assessments of comfort. Water loss was affected by diminished evaporation from the skin. Skin temperature sensitivity was changed and took many days after the dives to return to normal. Energy requirements increased for work purposes, but basal metabolic rate was undisturbed. Body chemistry altered at pressures in excess of 300 m s.w., for example thyroid hormone and nitrogen balances were affected. No decompression sickness was encountered until the pressures were low, but marked haematological changes could occur during decompression. Every change that occurred during these dives reverted to normal, mostly before the end of the decompression. It is concluded that diving with oxygen-helium breathing mixtures to depths as great as 540 m s.w. can be effective and safe. An attempt is made to assess the physiological significance of the principal findings.