RNA Junction Research Articles

Nucleic acid structure and recognition

We review the global structures adopted by branched nucleic acids, including three- and four-way helical junctions in DNA and RNA. We find that some general folding principles emerge. First, all the structures exhibit a tendency to undergo pairwise coaxial helical stacking when permitted by the local stereochemistry of strand exchange. Second, metal ions generally play an important role in facilitating folding of branched nucleic acids. These principles can be applied to functionally important branched nucleic acids, such as the Holliday DNA junction of genetic recombination, and the hammerhead ribozyme in RNA.

The RNA Subunit of Ribonuclease P from the Zebrafish, Danio rerio

A simple strategy has been devised to identify the gene encoding the RNA subunit of RNase P from the zebrafish, Danio rerio. The sequence obtained by amplification of genomic DNA with primers based on sequences common to two other vertebrates was confirmed by reverse transcription and amplification of RNA from a partially purified preparation of the holoenzyme. The 5' and 3' ends were determined by cyclizing the RNA, followed by reverse transcription and sequencing across the ligated RNA junction. The zebrafish sequence is 63% identical to that of Xenopus laevis nuclear RNase P RNA and 69% identical to the human RNase P RNA. A consensus secondary structure was constructed based on these nucleotide identities and on the many compensatory base changes in several regions among these three RNAs. The strategy used to obtain the zebrafish sequence should be useful in deriving analogous gene sequences from diverse classes of eukaryotes.

The global folding of four-way helical junctions in RNA, including that in U1 snRNA

Helical junctions are important elements in the architecture of folded RNA molecules. The global geometry of fully base-paired four-way junctions between RNA helices has been analyzed by comparative gel electrophoresis. Junctions appear to fold by pairwise coaxial helical stacking in one of two possible stereochemically equivalent isomers based upon alternative selections of stacking partners. In the presence of 1 mM Mg +, the two continuous helical axes are approximately at right angles to each other for all junctions studied, but the RNA junctions exhibit significant sequence-dependent differences in their structures as a function of ionic conditions. The four-way junction found in the U1 snRNA folded by coaxial helical stacking. It retained the 90° crossed stacked structure under all ionic conditions tested, despite the presence of a G · A mismatch at the point of strand exchange.

Novel regulation of pregnant human myometrial smooth muscle cell gap junctions by human chorionic gonadotropin.

Human myometrium contains hCG/LH receptors. There are fewer of these receptors during labor compared to no labor at preterm or term deliveries. Exogenous hCG can directly inhibit oxytocin-stimulated human myometrial contractions. These findings suggest that hCG may directly maintain myometrial quiescence during pregnancy. As maintenance of uterine quiescence may involve down-regulation of myometrial gap junctions, we investigated the effect of hCG on connexin-43 (CX-43) gene expression from RNA to protein and morphological gap junctions. The addition of 5 or 10 nM highly purified hCG to subconfluent cultures of pregnant myometrial smooth muscle cells resulted in a significant decrease in CX-43 protein levels. Higher hCG concentrations (100 and 1000 nM), however, had no effect. The maximal effect of hCG was seen at 4-8 h of culture, followed by recovery after a longer duration of culture. hCG treatment also concomitantly decreased CX-43 messenger RNA and morphological gap junctions. The hCG effect on CX-43 protein levels is hormone specific and mediated by protein kinase-A signaling. Estradiol and oxytocin increased, whereas progesterone decreased, CX-43 protein levels and morphological gap junctions. The oxytocin-induced increase was reversed by cotreatment with hCG. Although RU 486 alone had no effect on CX-43 protein levels, it prevented the down-regulating action of hCG and progesterone. In summary, our results demonstrate that hCG can directly decrease CX-43 messenger RNA, protein, and morphological gap junctions in cultured pregnant human myometrial smooth muscle cells. The hCG action is concentration and time dependent, hormone specific, and mediated by protein kinase-A signaling and appears to involve progesterone receptors. These data lend support to the concept that hCG could be one of the hormones responsible for maintaining uterine quiescence by down-regulating myometrial gap junctions during pregnancy.

Increased expression of connexin-43 in the rat myometrium during labor is associated with an increase in the plasma estrogen:progesterone ratio

The molecular mechanisms that regulate the synthesis of the myometrial gap junction protein, connexin-43 (Cx-43), are controversial. We measured myometrial Cx-43 messenger RNA, protein and gap junction frequency, and area in myometrial samples collected from nonpregnant rats and pregnant rats at days 5, 10, 15, 17, 18, 19, 20, 21, 22, 23 (during delivery), and 1 day postpartum and correlated these data with plasma concentrations of estradiol 17 beta and progesterone. Cx-43 transcripts were low or undetectable (connexin-43:glyceraldehyde phosphate dehydrogenase ratio < 0.2) in nonpregnant rats or in rats before day 10 of pregnancy. Transcripts rose to 0.52 +/- 0.11 on day 10, increased (2.9-fold) to 1.51 +/- 0.48 on day 22, and increased a further 2.9-fold to maximal levels of 4.42 +/- 0.67 during labor. Cx-43 protein was barely detectable on day 21 [0.12 +/- 0.04 relative optical density (ROD) units], increased 2.5-fold on day 22 (0.30 +/- 0.04 ROD units), and a further 3.7-fold during delivery (1.10 +/- 0.15 ROD units), at a time when gap junctions were present in large numbers in the cell membrane. Between day 21 and delivery the increase in Cx-43 transcripts (8.2-fold) and protein (9.2-fold) were of a similar magnitude. There was a significant positive correlation between the increases in Cx-43 transcripts and the increase in the ratio of plasma estradiol to progesterone. Levels of Cx-43 transcripts, protein, and gap junctions fell rapidly postpartum. Our data demonstrate: 1) that transcripts encoding the gap junction protein, Cx-43, are at maximal levels during delivery and that this increase is temporally associated with increases in Cx-43 protein and the appearance of gap junctions; and 2) that these data, in association with changes in plasma steroid concentrations, are consistent with myometrial Cx-43 transcript levels being regulated positively by estrogen and negatively by progesterone during pregnancy.

Increased expression of connexin-43 in the rat myometrium during labor is associated with an increase in the plasma estrogen:progesterone ratio.

The molecular mechanisms that regulate the synthesis of the myometrial gap junction protein, connexin-43 (Cx-43), are controversial. We measured myometrial Cx-43 messenger RNA, protein and gap junction frequency, and area in myometrial samples collected from nonpregnant rats and pregnant rats at days 5, 10, 15, 17, 18, 19, 20, 21, 22, 23 (during delivery), and 1 day postpartum and correlated these data with plasma concentrations of estradiol 17 beta and progesterone. Cx-43 transcripts were low or undetectable (connexin-43:glyceraldehyde phosphate dehydrogenase ratio < 0.2) in nonpregnant rats or in rats before day 10 of pregnancy. Transcripts rose to 0.52 +/- 0.11 on day 10, increased (2.9-fold) to 1.51 +/- 0.48 on day 22, and increased a further 2.9-fold to maximal levels of 4.42 +/- 0.67 during labor. Cx-43 protein was barely detectable on day 21 [0.12 +/- 0.04 relative optical density (ROD) units], increased 2.5-fold on day 22 (0.30 +/- 0.04 ROD units), and a further 3.7-fold during delivery (1.10 +/- 0.15 ROD units), at a time when gap junctions were present in large numbers in the cell membrane. Between day 21 and delivery the increase in Cx-43 transcripts (8.2-fold) and protein (9.2-fold) were of a similar magnitude. There was a significant positive correlation between the increases in Cx-43 transcripts and the increase in the ratio of plasma estradiol to progesterone. Levels of Cx-43 transcripts, protein, and gap junctions fell rapidly postpartum. Our data demonstrate: 1) that transcripts encoding the gap junction protein, Cx-43, are at maximal levels during delivery and that this increase is temporally associated with increases in Cx-43 protein and the appearance of gap junctions; and 2) that these data, in association with changes in plasma steroid concentrations, are consistent with myometrial Cx-43 transcript levels being regulated positively by estrogen and negatively by progesterone during pregnancy.

Alternative secondary structures in the 5' exon affect both forward and reverse self-splicing of the Tetrahymena intervening sequence RNA

The natural splice junction of the Tetrahymena large ribosomal RNA is flanked by hairpins that are phylogenetically conserved. The stem immediately preceding the splice junction involves nucleotides that also base pair with the internal guide sequence of the intervening sequence during splicing. Thus, precursors which contain wild-type exons can form two alternative helices. We have constructed a series of RNAs where the stem-loop in the 5' exon is more or less stable than in the wild-type precursor, and tested them in both forward and reverse self-splicing reactions. The presence of a stable hairpin in ligated exon substrates interferes with the ability of the intervening sequence to integrate at the splice junction. Similarly, the presence of the wild-type hairpin in the 5' exon reduces the rate of splicing 20-fold in short precursors. The data are consistent with a competition between unproductive formation of a hairpin in the 5' exon and productive pairing of the 5' exon with the internal guide sequence. The reduction of splicing by a hairpin that is a normal feature of rRNA structure is surprising; we propose that this attenuation is relieved in the natural splicing environment.

Recombination between satellite RNAs of turnip crinkle virus.

Turnip crinkle virus (TCV) is associated with satellite (sat) RNAs (sat-RNA D, sat-RNA F), defective interfering (DI) RNAs (DI RNA G, DI1 RNA), and one RNA with properties of both sat-RNAs and DI RNAs (sat-RNA C). When plants were inoculated with TCV, sat-RNA D and in vitro sat-RNA C transcripts containing non-viable mutations in the 5' domain, recombinant sat-RNAs were recovered. These recombinants were composed of sat-RNA D at the 5' end and sat-RNA C sequences at the 3' end. Analysis of 20 independent recombination junctions revealed that unequal crossing-over had occurred in planta in a region of sequence similarity between the two sat-RNAs which resulted in the duplication of 3-16 nucleotides. Thirty percent of the sat-RNA recombinants also had one to three additional nucleotides inserted at the crossover junctions which did not correspond to either sat-RNA C or sat-RNA D sequence. The right side of the recombination junctions always began with one of three consecutive nucleotides of sat-RNA C. Based on the similarity between this sequence of sat-RNA C, the right side junction of DI RNA G and the 5' end of TCV, as well as the sequence similarity between right side junctions of DI1 RNA and sat-RNA C and the 5' end of the sat-RNAs, a replicase-driven copy choice mechanism is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)

RNA mediated formation of a phosphorothioate diester bond

Previous results showed that multimeric, tandemly sequence-repeated forms of satellite tobacco ringspot virus RNA of the encapsidated polarity (STobRV (+)RNA) autolytically process at a specific phosphodiester bond, the junction. Substituting a phosphorothioate diester bond for the STobRV (+)RNA junction drastically slowed autolytic processing. Here we show that for the complementary STobRV (-)RNA, in contrast, replacing sets of phosphodiester bonds with phosphorothioate diester bonds, even at the junction, did not greatly slow autolytic processing or spontaneous ligation, the usual reactions of the unmodified RNA. In the ligation reaction STobRV (-)RNA directed the formation of an ApG phosphorothioate diester bond.

Two domains for splicing in the intron of the phage T4 thymidylate synthase ( td) gene established by nondirected mutagenesis

Of 97 nondirected T4 thymidylate synthase-defective ( td) mutations, 27 were mapped to the intron of the split td gene. Clustering of these intron mutations defined two domains that are functional in splicing, each within approximately 220 residues of the respective splice sites. Two selected mutations, tdN57 and tdN47, fell within phylogenetically conserved pairings, with tdN57 disrupting the exon I-internal guide pairing (P1) in the 5′ domain and tdN47 destabilizing the P9 helix in the 3′ domain. A splicing assay with synthetic oligonucleotides complementary to RNA junction sequences revealed processing defects for T4 tdN57 and T4 tdN47, both of which are impaired in cleavage at the 5′ and 3′ splice sites. Thus prokaryotic genetics facilitates association of specific residue changes with their consequences to splicing.

Human parainfluenza virus type 3: messenger RNAs, polypeptide coding assignments, intergenic sequences, and genetic map.

cDNA clones of mRNAs for the major nucleocapsid protein (NP), the nucleocapsid P protein plus the nonstructural C protein (P+C), and the matrix protein (M) of human parainfluenza virus type 3 (PF3) were identified by hybrid arrest and hybrid selection of in vitro translation. Previously, cDNA clones were identified and sequenced for the hemagglutinin-neuraminidase glycoprotein (HN) and the fusion glycoprotein (F) mRNAs (N. Elango, J. E. Coligan, R. C. Jambou, and S. Venkatesan, J. Virol. 57:481-489, 1986; M. K. Spriggs, R. A. Olmsted, S. Venkatesan, J. E. Coligan, and P. L. Collins, Virology 152:241-251, 1986). Synthetic oligonucleotides, designed from nucleotide sequences of the cDNAs, were used to direct dideoxynucleotide sequencing of gene junctions in PF3 genomic RNA (vRNA). From sequencing of vRNA, a sixth viral gene was detected and identified as the large nucleocapsid protein (L) gene by hybridization of a synthetic oligonucleotide to intracellular PF3 mRNAs separated by gel electrophoresis. The order of the six PF3 genes on vRNA was 3'-NP-P+C-M-F-HN-L-5'. The five intergenic regions consisted of the trinucleotide 3'-GAA. The PF3 genes initiated with semiconserved 10-nucleotide gene-start sequences and terminated with semiconserved 12-nucleotide gene-end sequences. The M gene terminated with an aberrant gene-end sequence; analysis of intracellular mRNA showed that this aberrant sequence correlated with a disproportionately high accumulation of readthrough mRNA. These studies showed that PF3 encodes six unique mRNAs (NP, P+C, M, F, HN, and L) that encode seven proteins (NP, P, C, M, F, HN, and L) and provided evidence of a close relationship between PF3 and Sendai (murine parainfluenza type 1) viruses.

Nucleotide sequence of the 26 S mRNA of the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus and deduced sequence of the encoded structural proteins.

A cDNA clone containing all of the 26 S mRNA coding region of the RNA genome of Venezuelan equine encephalitis (VEE) virus, virulent strain Trinidad donkey (TRD), has been constructed and sequenced. The nucleotide and deduced amino acid sequences of the 26 S RNA of VEE virus conform to the general organization of the alphavirus subgenomic mRNA. Excluding the poly(A) tail, the VEE 26 S RNA is 3913 nucleotides long with a protein coding region of 3762 nucleotides. Codon usage in the translated region is nonrandom and correlates well with that reported for Sindbis (SIN), Semliki Forest (SF), and Ross River (RR) alphaviruses. Highly conserved sequences of 19 to 22 nucleotides representing putative replicase recognition sites occur at the 26 S RNA junction region of the 42 S genomic RNA and at the 3' terminus immediately preceding the poly(A) tail. The conserved sequence at the 26 S/42 S junction region of VEE virus differs from that of other alphaviruses in that an ochre termination codon (UAA) is substituted for a GGU (Gly) codon present in the other viruses. The 5' and 3' noncoding regions (30 and 121 nucleotides, respectively) of the VEE 26 S RNA are shorter than has been reported for several other alphaviruses. The approximate transmembrane domains of the VEE E1 and E2 envelope glycoproteins have been identified. VEE E1 contains a single asparagine-linked glycosylation site, whereas E2 has three such sites, all of which are apparently glycosylated. The deduced amino acid sequence of the VEE polyprotein shows an overall homology of 44 to 46% with the precursor polyproteins of SIN, SF, and RR viruses. VEE virus capsid, E1, and E2 structural proteins show 43 to 46%, 50 to 53%, and 36 to 41% homology, respectively, with the cognate proteins of SIN, SF, and RR viruses.

Cloning and sequencing of 5' terminal sequences from avian infectious bronchitis virus genomic RNA.

The subgenomic RNAs of the fowl coronavirus infectious bronchitis virus (IBV) form a 3' co-terminal or 'nested' set. The presence of non-contiguous (leader) sequences fused to the 5' termini of murine hepatitis virus mRNAs has been demonstrated using RNase T1 oligonucleotide mapping and sequencing. The presence of a leader sequence on IBV mRNA A has been demonstrated previously. In this paper the presence of a leader identical to that present on the 5' terminus of IBV mRNA A is demonstrated to be present on the 5' terminus of IBV genomic RNA. This has been achieved by sequencing of primer extension products and cDNA clones containing the genomic leader. Analysis of these clones has revealed the presence of a sequence at the leader/genome-length RNA junction which is closely related to regions of homology identified previously within the genomic RNA sequence at the leader/body junctions of subgenomic RNAs. The implications of this finding for mechanisms of coronavirus RNA synthesis are discussed.

Time course of alfalfa mosaic virus RNA and coat protein synthesis in cowpea protoplasts

A study was made of the time course of the synthesis of viral plus-strand RNA, minus-strand RNA, and coat protein in alfalfa mosaic virus-infected Cowpea protoplasts. The three genomic RNAs were synthesized at different rates, as were their corresponding minus-strands. We conclude that viral RNA synthesis is regulated both at the level of minus-strand production and the level of plus-strand production. The synthesis of subgenomic RNA 4 was slower than that of its corresponding genomic RNA (RNA 3), indicating that an additional function, expressed later in infection, is required for production of subgenomic coat protein messenger. The data support a model for RNA 4 synthesis involving internal initiation by the RNA polymerase at the intercistronic junction in minus-strand RNA 3. The temporal relationship of the synthesis of RNA 3, RNA 4, and coat protein is discussed.

The use of a heptadeoxyribonucleotide as a specific primer for prolactin mRNA: A prediction of ambiguous RNA splicing

A 5′-labelled oligonucleotide, d(T-C-A-T-C-A-T), complementary to the mRNA sequence coding for the amino acid sequence (Met·Met) present in preprolactin was used to initiate the synthesis of a 135 nucleotide cDNA from poly(A) +RNA isolated from the rat anterior pituitary. Nucleotide sequence analysis of the cDNA revealed that it corresponded to a reverse transcript of rat prolactin mRNA. These results established that an oligonucleotide as short as a heptamer with a single nucleotide mismatch at its 5′ end can be used to obtain a specific cDNA which can be sequenced directly. The oligonucleotide initiated cDNA sequence and the sequence of a cloned cDNA to rat prolactin mRNA [Cooke, N., et al. , J. Biol. Chem. 255 , 6502 (1980)] differ in that the former sequence has an additional codon (GCA) for alanine at a position corresponding to the tenth amino acid of the signal sequence of rat preprolactin. This result could be explained by two populations of rat prolactin mRNA arising from ambiguous splicing at an exon-intron junction in the precursor RNA.

Structure of the 5.8S RNA component of the 5.8S-28S ribosomal RNA junction complex.

Structure of the 5.8S RNA component of the 5.8S-28S ribosomal RNA junction complex.