The spatial configuration of ordered polynucleotide chains. II. The poly(rA) helix.
(57/3703)
Approximate details of the spatial configuration of the ordered single-stranded poly(rA) molecule in dilute solution have been obtained in a combined theoretical analysis of base stacking and chain flexibility. Only those regularly repeating structures which fulfill the criterion of conformational flexibility (based upon all available experimental and theoretical evidence of preferred bond rotations) and which also exhibit the right-handed base stacking pattern observed in nmr investigations of poly(rA) are deemed suitable single-stranded helices. In addition, the helical geometry of the stacked structures is required to be consistent with the experimentally observed dimensions of both completely ordered and partially ordered poly(rA) chains. Only a single category of poly(rA) helices (very similar in all conformational details to the individual chains of the poly(rA) double-stranded X-ray structure) is thus obtained. Other conformationally feasible polynucleotide helices characterized simply by a parallel and overlapping base stacking arrangement are also discussed. (+info)
Spatially regulated translation in embryos: asymmetric expression of maternal Wnt-11 along the dorsal-ventral axis in Xenopus.
(58/3703)
Transition from symmetry to asymmetry is a central theme in cell and developmental biology. In Xenopus embryos, dorsal-ventral asymmetry is initiated by a microtubule-dependent cytoplasmic rotation during the first cell cycle after fertilization. Here we show that the cytoplasmic rotation initiates differential cytoplasmic polyadenylation of maternal Xwnt-11 RNA, encoding a member of the Wnt family of cell-cell signaling factors. Translational regulation of Xwnt-11 mRNA along the dorsal-ventral axis results in asymmetric accumulation of Xwnt-11 protein. These results demonstrate spatially regulated translation of a maternal cell-signaling factor along the vertebrate dorsal-ventral axis and represent a novel mechanism for Wnt gene regulation. Spatial regulation of maternal RNA translation, which has been established in invertebrates, appears to be an evolutionarily conserved mechanism in the generation of intracellular asymmetry and the consequential formation of the multicellular body pattern. (+info)
Translational control of dosage compensation in Drosophila by Sex-lethal: cooperative silencing via the 5' and 3' UTRs of msl-2 mRNA is independent of the poly(A) tail.
(59/3703)
Translational repression of male-specific-lethal 2 (msl-2) mRNA by Sex-lethal (SXL) controls dosage compensation in Drosophila. In vivo regulation involves cooperativity between SXL-binding sites in the 5' and 3' untranslated regions (UTRs). To investigate the mechanism of msl-2 translational control, we have developed a novel cell-free translation system from Drosophila embryos that recapitulates the critical features of mRNA translation in eukaryotes: cap and poly(A) tail dependence. Importantly, tight regulation of msl-2 translation in this system requires cooperation between the SXL-binding sites in both the 5' and 3' UTRs, as seen in vivo. However, in contrast to numerous other developmentally regulated mRNAs, the regulation of msl-2 mRNA occurs by a poly(A) tail-independent mechanism. The approach described here allows mechanistic analysis of translational control in early Drosophila development and has revealed insights into the regulation of dosage compensation by SXL. (+info)
Intercalation of proflavine and a platinum derivative of proflavine into double-helical Poly(A).
(60/3703)
The equilibria and kinetics of the interactions of proflavine (PR) and its platinum-containing derivative [PtCl(tmen)(2)HNC(13)H(7)(NHCH(2)CH(2))(2)](+) (PRPt) with double-stranded poly(A) have been investigated by spectrophotometry and Joule temperature-jump relaxation at ionic strength 0.1 M, 25 degrees C, and pH 5.2. Spectrophotometric measurements indicate that base-dye interactions are prevailing. T-jump experiments with polarized light showed that effects due to field-induced alignment could be neglected. Both of the investigated systems display two relaxation effects. The kinetic features of the reaction are discussed in terms of a two-step series mechanism in which a precursor complex DS(I) is formed in the fast step, which is then converted to a final complex in the slow step. The rate constants of the fast step are k(1) = (2.5 +/- 0.4) x 10(6) M(-1) s(-1), k(-1) = (2.4 +/- 0.1) x 10(3) s(-1) for poly(A)-PR and k(1) = (2.3 +/- 0.1) x 10(6) M(-1) s(-1), k(-1) = (1.6 +/- 0.2) x 10(3) s(-1) for poly(A)-PRPt. The rate constants for the slow step are k(2) = (4.5 +/- 0.5) x 10(2) s(-1), k(-2) = (1.7 +/- 0.1) x 10(2) s(-1) for poly(A)-PR and k(2) = 9.7 +/- 1.2 s(-1), k(-2) = 10.6 +/- 0.2 s(-1) for poly(A)-PRPt. Spectrophotometric measurements yield for the equilibrium constants and site size the values K = (4.5 +/- 0.1) x 10(3) M(-1), n = 1.3 +/- 0.5 for poly(A)-PR and K = (2.9 +/- 0.1) x 10(3) M(-1), n = 2.3 +/- 0.6 for poly(A)-PRPt. The values of k(1) are similar and lower than expected for diffusion-limited reactions. The values of k(-1) are similar as well. It is suggested that the formation of DS(I) involves only the proflavine residues in both systems. In contrast, the values of k(2) and k(-2) in poly(A)-PRPt are much lower than in poly(A)-PR. The results suggest that in the complex DS(II) of poly(A)-PRPt both proflavine and platinum residues are intercalated. In addition, a very slow process was detected and ascribed to the covalent binding of Pt(II) to the adenine. (+info)
Somatic sequence variation at the Friedreich ataxia locus includes complete contraction of the expanded GAA triplet repeat, significant length variation in serially passaged lymphoblasts and enhanced mutagenesis in the flanking sequence.
(61/3703)
The vast majority of Friedreich ataxia patients are homozygous for large GAA triplet repeat expansions in intron 1 of the X25 gene. Instability of the expanded GAA repeat was examined in 23 chromosomes bearing 97-1250 triplets in lymphoblastoid cell lines passaged 20-39 times. Southern analyses revealed 18 events of significant changes in length ranging from 69 to 633 triplets, wherein the de novo allele gradually replaced the original over 1-6 passages. Contractions and expansions occurred with equal frequency and magnitude. This behavior is unique in comparison with other large, non-coding triplet repeat expansions [(CGG)(n)and (CTG)(n)] which remain relatively stable under similar conditions. We also report a rare patient who, having inherited two expanded alleles, showed evidence of contracted GAA repeats ranging from nine to 29 triplets in DNA from two independent peripheral blood samples. The GAA triplet repeat is known to adopt a triplex structure, and triplexes in transcribed templates cause enhanced mutagenesis. The poly(A) tract and a 135 bp sequence, both situated immediately upstream of the GAA triplet repeat, were therefore examined for somatic mutations. The poly(A) tract showed enhanced instability when in cis with the GAA expansion. The 135 bp upstream sequence was found to harbor a 3-fold excess of point mutations in DNA derived from individuals homozygous for the GAA triplet repeat expansion compared with normal controls. These data are likely to have important mechanistic and clinical implications. (+info)
c-mos and cdc2 cooperate in the translational activation of fibroblast growth factor receptor-1 during Xenopus oocyte maturation.
(62/3703)
During oocyte maturation in Xenopus, previously quiescent maternal mRNAs are translationally activated at specific times. We hypothesized that the translational recruitment of individual messages is triggered by particular cellular events and investigated the potential for known effectors of the meiotic cell cycle to activate the translation of the FGF receptor-1 (XFGFR) maternal mRNA. We found that both c-mos and cdc2 activate the translation of XFGFR. However, although oocytes matured by injection of recombinant cdc2/cyclin B translate normal levels of XFGFR protein, c-mos depletion reduces the level of XFGFR protein induced by cdc2/cyclin B injection. In oocytes blocked for cdc2 activity, injection of mos RNA induced low levels of XFGFR protein, independent of MAPK activity. Through the use of injected reporter RNAs, we show that the XFGFR 3' untranslated region inhibitory element is completely derepressed by cdc2 alone. In addition, we identified a new inhibitory element through which both mos and cdc2 activate translation. We found that cdc2 derepresses translation in the absence of polyadenylation, whereas mos requires poly(A) extension to activate XFGFR translation. Our results demonstrate that mos and cdc2, in addition to functioning as key regulators of the meiotic cell cycle, cooperate in the translational activation of a specific maternal mRNA during oocyte maturation. (+info)
In silico detection of control signals: mRNA 3'-end-processing sequences in diverse species.
(63/3703)
We have investigated mRNA 3'-end-processing signals in each of six eukaryotic species (yeast, rice, arabidopsis, fruitfly, mouse, and human) through the analysis of more than 20,000 3'-expressed sequence tags. The use and conservation of the canonical AAUAAA element vary widely among the six species and are especially weak in plants and yeast. Even in the animal species, the AAUAAA signal does not appear to be as universal as indicated by previous studies. The abundance of single-base variants of AAUAAA correlates with their measured processing efficiencies. As found previously, the plant polyadenylation signals are more similar to those of yeast than to those of animals, with both common content and arrangement of the signal elements. In all species examined, the complete polyadenylation signal appears to consist of an aggregate of multiple elements. In light of these and previous results, we present a broadened concept of 3'-end-processing signals in which no single exact sequence element is universally required for processing. Rather, the total efficiency is a function of all elements and, importantly, an inefficient word in one element can be compensated for by strong words in other elements. These complex patterns indicate that effective tools to identify 3'-end-processing signals will require more than consensus sequence identification. (+info)
Different positioning elements select poly(A) sites at the 3'-end of GCN4 mRNA in the yeast Saccharomyces cerevisiae.
(64/3703)
Cleavage and polyadenylation of eukaryotic mRNA requires efficiency and positioning elements in the 3'-untranslated region (3'-UTR) of the mRNA. Specific point mutations were introduced into the yeast GCN4 3'-UTR to detect sequence motifs which are involved in the positioning of the poly(A) site. 3'-End proces-sing activities of different GCN4 3'-UTR alleles were measured in an in vivo test system. Point mutations in an AAGAA motif defocussed selection of the poly(A) sites of the GCN4 3'-UTR to various additional poly(A) sites instead of the single site of the wild-type GCN4 3'-UTR. A strain with an intact wild-type GCN4 3'-UTR but impaired in RNA15 encoding an RNA-binding processing factor showed a similar defocussed pattern of poly(A) site selection. Remarkably, two additional sequence motifs upstream of the AAGAA motif which resemble yeast efficiency motifs independently affected poly(A) site positioning but not efficiency of 3'-end processing. Mutations in one motif resulted in an additional upstream poly(A) site. Alterations of the other motif shifted the poly(A) sites exclusively to two downstream poly(A) sites. These data suggest several contact points between the precursor mRNA and the polyadenylation machinery in yeast. (+info)