Trans insertion-splicing: ribozyme-catalyzed insertion of targeted sequences into RNAs

A group I intron-derived ribozyme from Pneumocystis carinii has been previously shown to bind an exogenous RNA substrate, splice out an internal segment, and then ligate the two ends back together (the trans excision-splicing reaction). We demonstrate that this same ribozyme can perform a trans insertion-splicing (TIS) reaction, where the ribozyme binds two exogenous RNA substrates and inserts one directly into the other. Reactions were optimized for both yield and rate, with optimum reactions carried out in 10 mM MgCl(2) for 2 h. Reaction products are stable, with no visible loss at extended times. The ribozyme recognizes the two substrates primarily through base pairing and requires an omegaG on the ribozyme and an omegaG on the sequence being inserted. We give evidence that the reaction mechanism is not the reverse of the trans excision-splicing reaction, but is composed of three steps, with intermediates attached to the ribozyme. Surprisingly, the internal guide sequence of the ribozyme is utilized to sequentially bind both substrates, forming independent P1 helices. This is an indication that ribozymes with essentially the native intron sequence can catalyze reactions significantly more dynamic and complex than self-splicing. The implications of group I intron-derived ribozymes being able to catalyze this unique reaction, and via this mechanism, are discussed.     

Ribozyme-catalyzed aminoacylation from CoA thioesters

Coenzyme A (CoA) thioesters play essential roles in modern metabolism. To demonstrate plausible biochemical functions of thioesters in the RNA world, we have isolated a new class of ribozymes (ACT) that catalyze self-aminoacylation from a number of CoA thioesters with catalytic efficiencies ranging from 7000 to 24 000 M(-1) x min(-1). Active thioester substrates are required to contain both a free alpha-amino group in the acyl moiety and a CoA as the thiol component. We hypothesize ribozyme-based aminoacylation systems using aminoacyl thioesters of CoA as the ancestors of modern aminoacyl tRNA synthetases. On the basis of our previous results [Huang et al. (2000) Biochemistry 39, 15548-15555; Coleman and Huang (2002) Chem. Biol. 9, 1227-1236], an extensive RNA-catalyzed "metabolic pathway" involving CoA and its thioesters is proposed. Complex contemporary metabolic systems could have evolved from the proposed ribozyme pathways.     

Selective cleavage of bcr-abl chimeric RNAs by a ribozyme targeted to non-contiguous sequences.

Conventionally designed ribozymes may be unable to cleave RNA at sites which are inaccessible due to secondary structure. In addition, it may also be difficult to specifically target a conventionally designed ribozyme to some chimeric RNA molecules. Novel approaches for ribozyme targeting were developed by using the L6 bcr-abl fusion RNA as a model. Using one approach, we successfully directed ribozyme nucleation to a site on the bcr-abl RNA that is distant from the GUA cleavage site. These ribozymes bound to the L6 substrate RNA via an anchor sequence that was complementary to bcr sequences. The anchor was necessary for efficient cleavage as the anchor minus ribozyme, a conventionally designed ribozyme, was inefficient at catalyzing cleavage at this same site. The effect of anchor sequences on catalytic rates was determined for two of these ribozymes. Ribozymes generated by a second approach were designed to cleave at a CUU site in proximity to the bcr-abl junction. Both approaches have led to the development of a series of ribozymes specific for both the L6 and K28 bcr-abl chimeric RNAs, but not normal abl or bcr RNAs. The specificity of the ribozyme correlated in part with the ability of the ribozyme to bind substrate as demonstrated by gel shift analyses. Secondary structure predictions for the RNA substrate support the experimental results and may prove useful as a theoretical basis for the design of ribozymes.     

Non-random frequencies of short oligonucleotide sequences within poliomyelitis and encephalomyocarditis virus RNAs.

In poliomyelitis and encephalomyocarditis virus RNAs the relative frequencies of several short oligonucleotide sequences differ substantially from those expected on a random basis. The frequencies of CpGp-containing sequences are low, and the frequency of GpUpApGp is considerably lower than that of GpApUpGp. The relationship of these findings to those obtained from host cell RNAs is descussed.     

The nucleotide sequences of 5S ribosomal RNAs from four Bryophyta-species

The nucleotide sequences of cytoplasmic 5S rRNA from four bryophytes, Marchantia polymorpha, Lophocolea heterophylla, Plagiomnium trichomanes and Anthoceros punctatus have been determined. These RNAs are 119 nucleotides long except for the Anthoceros RNA that has 118 nucleotides. Their sequences are highly similar to each other (91-99% identity) and are more related to those from seed plants (78-83% identity) than to those from green algae (61-73% identity).     

The excision of intervening sequences from Salmonella 23S ribosomal RNA

Novel, approximately 90 bp intervening sequences (IVs) were discovered within the 23S rRNA genes of S. typhimurium and S. arizonae. These non-rRNA sequences are transcribed and then excised during rRNA maturation. The rRNA fragments that result from the excision of the extra sequences are not religated. This results in fragmented 23S rRNAs. The excision of one IVS was shown to be catalyzed in vivo and in vitro by ribonuclease III. These IVSs are highly volatile evolutionarily, sometimes occurring in only some of the multiple rRNA operons of a particular cell. The sporadic nature of the occurrence of fragmented rRNAs among closely related organisms argues that such fragmentation is a derived state, not a primitive one. Possible sources of these IVSs, their parallels with internal transcribed spacers and introns in eukaryotes, and their possible roles in the evolutionary process are discussed.     

Structure and metabolism of adenovirus RNAs containing sequences from the fiber gene

The body of adenovirus fiber messenger RNA is specified by viral r-strand co-ordinates 86.2 to 91.2. Since this mRNA is transcribed from the major late promoter at map position 16, nuclear precursors to the mRNA could be as large as 84% of the length of the 35,000 nucleotide genome. This study identified and characterized polyadenylated nuclear RNAs that contain fiber sequences and therefore are possible processing intermediates. These nuclear RNAs were characterized by hybridization of [³H]RNA preparations and by electron microscopy of RNA-DNA hybrids. Three size classes of RNAs containing fiber sequences were identified: (1) a 22 S species maps from 86.2 to 90.3. This RNA has essentially the same co-ordinates as fiber mRNA. (2) Two 28 S species have co-ordinates of 80.1 to 90.4 and 85.9 to 96.9, respectively. Thus one species has a 5′ terminus coincident with that of the mRNA body, and one has a 3′ terminus coincident with that of the 3′ end of the mRNA body. The polyadenylated terminus at 96.9 does not coincide with the 3′ end of any known mRNA. (3) There are at least two 35 S species. The 3′ end of one species is coincident with that of fiber mRNA. The 3′ terminus of the second RNA is at approximately 96.9.The labeling kinetics of each of these polyadenylated nuclear RNAs were investigated. In continuous label experiments, the two 35 S RNAs and the 85.9 to 96.9 28 S RNA became uniformly labeled in approximately 60 minutes. The 22 S RNA and the 80.1 to 90.4 28 S species continued to accumulate for at least several hours. These results are consistent with a precursor function for the 35 S RNAs and the 85.9 to 96.9 28 S species. The structures of the putative precursors imply that processing of the 3′ end is not a prerequisite for 5′ cleavage.     

The nucleotide sequences of the initiator transfer RNAs from bean cytoplasm and chloroplasts

The initiator tRNAsMet from the cytoplasm and chloroplasts of Phaseolus vulgaris have been purified and sequenced. The sequence of bean cytoplasmic initiator tRNAiMet is : pA-U-C-A-G-A-G-U-m1G-m2G-C-G-C-A-G-C-G-G-A-A-G-C-G-U-m2G-G-U-G-G-G2-C-C-C-A-U-t6A-A-C-C-C-A-C-A-G-m7G-D-m5C-C-C-A-G-G-A-psi-C-G-m1A-A-A-C-C-U-Gm-G-C-U-C-U-G-A-U-A-C-C-AOH. The sequence of bean cytoplasmic tRNAiMet is almost identical to that of wheat germ and shows a high degree of homology with other cytoplasmic initiator tRNAs. The sequence of bean chloroplast initiator tRNAfMet is : pC-G-C-G-G-A-G-U-A-G-A-G-C-A-A-C-U-U-Gm-G-D-A-G-C-U-C-G-C-A-A-G-G-C-U-C-A-U-A-A-C-C-U-U-G-A-A-m7G-acp3U-U-A-C-G-G-G-T-psi-C-A-A-A-U-C-C-C-G-U-C-U-C-C-G-C-A-A- C-C-AOH. Bean chloroplast initiator tRNAfMet sequence shows procaryotic characteristics at the 5' end of the acceptor stem and in the TpsiC loop, but also contains some distinctive features.     

Retina-specific gene excision by targeted expression of Cre recombinase

The use of Cre recombinase for conditional targeting permits the controlled removal or activation of genes in specific tissues and at specific times of development. The Rho–Cre mice provide an improved tool for studying gene ablation in rod photoreceptor cells. To establish a robust expression of Rho–Cre transgenic mice that would be useful for the study of various protein functions in photoreceptor cells, a total 11,987 kb fragment (pNCHS4 Rho–NLS–cre) containing human rhodopsin promoter was cloned. The Rho–Cre plasmid was digested with EcoR1 and I Ceu-1, and the 9.316 kb fragment containing the hRho promoter and Cre recombinase gel was purified. To generate transgenic mice, the purified DNA fragment was injected into fertilized oocytes according to standard protocols. ROSA26R reported the steady expression of Rho–Cre especially in photoreceptor cells, allowing further excising proteins in rod photoreceptors across the retina. This Rho–Cre transgenic line should thus prove useful as a general deletor line for genetic analysis of diverse aspects of retinopathy.     

Spermatocyte-specific gene excision by targeted expression of Cre recombinase

Transgenic mice carrying the coding sequence of the Cre recombinase, whose expression was driven by the spermatocyte-specific Pgk-2 promoter, were generated. These mice were crossed with a reporter transgenic line, which produces beta-galactosidase depending on the occurrence of loxP-mediated DNA recombination. When DNA of the offspring was analyzed by PCR and Southern blotting, signals that appear after the recombination were detectable only in the testis. Histochemical analyses revealed that beta-galactosidase was present in spermatocytes and spermatogenic cells at later differentiation stages. However, the distribution of the protein was not uniform in all spermatocytes. Analyses of genomic DNA of the next generation indicated that recombination took place in about 70% of spermatogenic cells. From these results, we concluded that this transgenic line possessing Pgk-2-driven expression of the Cre recombinase should be useful for identifying spermatogenic genes that function at or after the spermatocyte stage.     

Precise excision of intervening sequences from precursor tRNAs by a membrane-associated yeast endonuclease

Splicing of transfer RNA precursors containing intervening sequences proceeds in two distinct stages: endonucleolytic cleavage, followed by ligation. We have physically separated endonuclease and ligase activities from extracts of yeast cells, and we report properties of the partially purified endonuclease preparation. The endonuclease behaves as an integral membrane protein: it is purified from a membrane fraction from which it can be solubilized with nonionic detergents, and the activity of the endonuclease in the membrane fraction is stimulated by nonionic detergents. The endonuclease cleaves precursor tRNAs at two sites to excise the intervening sequence precisely. Both the extent and the accuracy of cleavage are enhanced by the presence of spermidine; the degree of stimulation varies with the pre-tRNA substrate. The cleavage products possess 5'-hydroxyl and 2',3'-cyclic phosphodiester termini. The cyclic phosphodiester termini can be opened to 2'-phosphates by a cyclic phosphodiesterase activity in the preparation.     

Micro RNAs: tiny sequences with enormous potential

The liver plays a central role in glucose homeostasis in the whole-body by responding to environmental factors including nutrients, hormones, and oxygen. In conditions of metabolic overload such as diabetes mellitus and obesity, coordinated regulation between oxygen supply and consumption has been reported to be disrupted and subsequently cause tissue hypoxia, although pathological significance of the disease-related hypoxia remains elusive. To investigate the role of tissue hypoxia in the liver on systemic glucose homeostasis, mice lacking HIF-1α gene, a critical component of a master regulator of hypoxic response, in hepatocytes were exposed to high fat/sucrose diet (HFSD). Exposure to HFSD for 5 weeks elicited liver hypoxia with a transient increase in HIF-1α protein expression in the liver of control mice. Glucose disposal was marginally impaired in control mice when challenged oral glucose tolerance test, but such impairment was enhanced in the mutant mice. This alteration was accompanied by a complete inhibition of glucokinase induction with a significant reduction of hepatic glucose uptake. Mice fed HFSD for 20 weeks exhibited fasting hyperglycemia and glucose intolerance, whereas these metabolic phenotypes deteriorated considerably with severe insulin resistance in skeletal muscles and adipose tissues in the mutant mice. These findings suggest that HIF-1 in hepatocytes plays protective roles against the progression of diabetes mellitus.     

Nucleotide sequences of nuclear U1A RNAs from chicken, rat and man.

The methods of enzymatic and chemical treatment of end-labeled RNA were applied to the determination of the nucleotide sequence of chicken and man U1A RNA and to the reexamination of that of rat U1A RNA. The chemical method allowed the easy demonstration of the cap structure. All three RNA were 165 nucleotide long. Two hitherto non described modified pyrimidines were detected close to the 5' end. Only 9 base substitutions were observed from chicken to man indicating high degree of conservation of U1A RNA through evolution.     

Complementarity of sequences in low molecular weight RNAs to regions of messenger and ribosomal RNAs.

Total low molecular weight nuclear RNAs of mouse ascites cells have been labeled in vitro and used as probes to search for complementary sequences contained in nuclear or cytoplasmic RNA. From a subset of hybridizing lmw RNAs, two major species of 58,000 and 35,000 mol. wt. have been identified as mouse 5 and 5.8S ribosomal RNA. Mouse 5 and 5.8S rRNA hybridize not only to 18 and 28S rRNA, respectively, but also to nuclear and cytoplasmic poly(A+) RNA. Northern blot analysis and oligo-dT cellulose chromatography have confirmed the intermolecular base-pairing of these two small rRNA sequences to total poly(A+) RNA as well as to purified rabbit globin mRNA. 5 and 5.8S rRNA also hybridize with positive (coding) but not negative (noncoding) strands of viral RNA. Temperature melting experiments have demonstrated that their hybrid stability with mRNA sequences is comparable to that observed for the 5S:18S and 5.8S:28S hybrids. The functional significance of 5 and 5.8S rRNA base-pairing with mRNAs and larger rRNAs is unknown, but these interactions could play important coordinating roles in ribosome structure, subunit interaction, and mRNA binding during translation.