Under-carboxylated osteocalcin regulates glucose and lipid metabolism during pregnancy and lactation in rats.

PURPOSE: Under-carboxylated osteocalcin (UcOC), a bone-released hormone is suggested to regulate energy metabolism. Pregnancy and lactation physiological conditions that require high levels of energy. The current study attempts to examine whether UcOC is involved in regulating energy metabolism during these conditions using adult Wistar rats. METHODS AND RESULTS: Insulin tolerance tests indicated insulin resistance during late pregnancy (day 19 of pregnancy; P19) and insulin sensitivity during early lactation (day 6 of lactation; L6). Gene expression analyses suggested that muscle glucose metabolism was downregulated during P19 and enhanced during L6. Concomitantly, circulatory UcOC levels were lower during pregnancy but higher during early lactation; the rise in UcOC levels was tightly linked to the lactation process. Altering endogenous UcOC levels pharmacologically with warfarin and alendronate in P19 and L6 rats changed whole-body insulin response and muscle glucose transporter (Glut4) expression. Glut4 expression can be increased by either UcOC or estrogen receptors (ERs), both of which act independent of each other. A high fat diet decreased UcOC levels and insulin sensitivity in lactating rats, suggesting that diet can compromise UcOC-established energy homeostasis. Gene expression of lipid metabolism markers and triglyceride levels suggested that UcOC suppression during early pregnancy is an essential step in maternal lipid storage. CONCLUSION: Taken together, we found that UcOC plays an important role in energy homeostasis via regulation of glucose and lipid metabolism during pregnancy and lactation.

Trans-splicing into highly abundant albumin transcripts for production of therapeutic proteins in vivo.

Spliceosome-mediated RNA trans-splicing has emerged as an exciting mode of RNA therapy. Here we describe a novel trans-splicing strategy, which targets highly abundant pre-mRNAs, to produce therapeutic proteins in vivo. First, we used a pre-trans-splicing molecule (PTM) that mediated trans-splicing of human apolipoprotein A-I (hapoA-I) into the highly abundant mouse albumin exon 1. Hydrodynamic tail vein injection of the hapoA-I PTM plasmid in mice followed by analysis of the chimeric transcripts and protein, confirmed accurate and efficient trans-splicing into albumin pre-mRNA and production of hapoA-I protein. The versatility of this approach was demonstrated by producing functional human papillomavirus type-16 E7 (HPV16-E7) single-chain antibody in C57BL/6 mice and functional factor VIII (FVIII) and phenotypic correction in hemophilia A mice. Altogether, these studies demonstrate that trans-splicing to highly abundant albumin transcripts can be used as a general platform to produce therapeutic proteins in vivo.

A generalizable strategy for imaging pre-mRNA levels in living subjects using spliceosome-mediated RNA trans-splicing.

UNLABELLED: Molecular imaging of gene expression is currently hindered by the lack of a generalizable platform for probe design. For any gene of interest, a probe that targets protein levels must often be generated empirically. Targeting gene expression at the level of mRNA, however, would allow probes to be built on the basis of sequence information alone. Presented here is a class of generalizable probes that can image pre-mRNA in a sequence-specific manner, using signal amplification and a facile method of delivery. METHODS: Pre-trans-splicing molecules (PTMs) were engineered to capitalize on the phenomenon of spliceosome-mediated RNA trans-splicing. Using a modular binding domain that confers specificity by base-pair complementarity to the target pre-mRNA, PTMs were designed to target a chimeric target mini gene and trans-splice the Renilla luciferase gene onto the end of the target. PTMs and target genes were transfected in cell culture and assessed by luciferase assay, reverse-transcriptase polymerase chain reaction, Western blot, and rapid analysis of 5' cDNA ends. PTMs and target genes were also assessed in vivo by hydrodynamic delivery in mice. RESULTS: Efficiency and specificity of the trans-splicing reaction were found to vary depending on the binding domain length and structure. Specific trans-splicing was observed in living animals (P = 0.0862, Kruskal-Wallis test). CONCLUSION: Described here is a model system used to demonstrate the feasibility of spliceosome-mediated RNA trans-splicing for imaging gene expression at the level of pre-mRNA using optical imaging techniques in living animals. The experiments reported here show proof of principle for a generalizable imaging probe against RNA that can amplify signal on detection and be delivered using existing gene delivery methodology.

Correction of DNA protein kinase deficiency by spliceosome-mediated RNA trans-splicing and sleeping beauty transposon delivery.

Spliceosome-mediated RNA trans-splicing (SMaRT) is an emerging technology for the repair of defective pre-messenger RNA (pre-mRNA) molecules. It is especially useful in the treatment of genetic disorders involving large genes. Although viral vectors have been used for achieving long-lasting expression of trans-splicing molecules, the immunogenicity and suboptimal safety profiles associated with viral-based components could limit the widespread application of SMaRT in the repair of genetic defects. Here, we tested whether the non-viral Sleeping Beauty (SB) transposon system could mediate stable delivery of trans-splicing molecules designed to correct the genetic defect responsible for severe combined immune deficiency (SCID). This immunological disorder is caused by a point mutation within the 12.4 kilobase (kb) gene encoding the DNA protein kinase catalytic subunit (DNA-PKcs) and is associated with aberrant DNA repair, defective T- and B-cell production, and hypersensitivity to radiation-induced injury. Using a novel SB-based trans-splicing vector, we demonstrate stable mRNA correction, proper DNA-PKcs protein production, and conference of a radiation-resistant phenotype in a T-cell thymoma cell line and SCID multipotent adult progenitor cells (MAPCs). These results suggest that SB-based trans-splicing vectors should prove useful in facilitating the correction of endogenous mutated mRNA transcripts, including the DNA-PKcs defect present in SCID cells.

Molecular imaging of gene expression in living subjects by spliceosome-mediated RNA trans-splicing.

Spliceosome-mediated RNA trans-splicing (SMaRT) provides an effective means to reprogram mRNAs and the proteins they encode. SMaRT technology has a broad range of applications, including RNA repair and molecular imaging, each governed by the nature of the sequences delivered by the pre-trans-splicing molecule. Here, we show the ability of SMaRT to optically image the expression of an exogenous gene at the level of pre-mRNA splicing in cells and living animals. Because of the modular design of pre-trans-splicing molecules, there is great potential to employ SMaRT to image the expression of any arbitrary gene of interest in living subjects. In this report, we describe a model system that demonstrates the feasibility of imaging gene expression by transsplicing in small animals. This represents a previously undescribed approach to molecular imaging of mRNA levels in living subjects.

5' exon replacement and repair by spliceosome-mediated RNA trans-splicing.

Spliceosome-mediated RNA trans-splicing (SMaRT) has been used previously to reprogram mutant endogenous CFTR and factor VIII mRNAs in human epithelial cell and tissue models and knockout mice, respectively. Those studies used 3' exon replacement (3'ER); a process in which the distal portion of RNA is reprogrammed. Here, we also show that the 5' end of mRNA can be completely rewritten by 5'ER. For proof-of-concept, and to test whether 5'ER could generate functional CFTR, we generated a mutant minigene target containing CFTR exons 10-24 (deltaF508) and a mini-intron 10, and a pretrans-splicing molecule (targeted to intron 10) containing CFTR exons 1-10 (+F508), and tested these two constructs in 293T cells for anion efflux transport. Cells cotransfected with target and PTM showed a consistent increase in anion efflux, but there was no response in control cells that received PTM or target alone. Using a LacZ reporter system to accurately quantify trans-splicing efficiency, we tested several unique PTM designs. These studies provided two important findings as follows: (1) efficient trans-splicing can be achieved by binding the PTM to different locations in the target, and (2) relatively few changes in PTM design can have a profound impact on trans-splicing activity. Tethering the PTM close to the target 3' splice site (as opposed to the donor site) and inserting an intron in the PTM coding resulted in a 65-fold enhancement of LacZ activity. These studies demonstrate that (1) SMaRT can be used to reprogram the 5' end of mRNA, and (2) efficiency can be improved substantially.

Development of spliceosome-mediated RNA trans-splicing (SMaRT) for the correction of inherited skin diseases.

Gene therapy of large genes (e.g. plectin and collagen genes) is hampered by size limitations for insertions of the currently used viral vectors. To reduce the size of these insertions spliceosome-mediated RNA trans-splicing (SMaRT), which provides intron-specific gene-correction at the pre-RNA level, can be an alternative approach. To test its applicability in skin gene therapy, SMaRT was used in the context of the 4003delTC mutation in the collagen XVII gene (COL17A1) causing generalized atrophic benign junctional epidermolysis bullosa. A beta-galactosidase (beta-gal) trans-splicing assay system was established using intron 51 of COL17A1 as the target for trans-splicing. In this system, intron 51 is flanked by the 5'exon and the 3'exon of the beta-gal gene, the latter containing two in-frame stop codons. Cotransfection of a pre-trans-splicing molecule consisting of the binding domain of intron 51 and the 3'exon of beta-gal without the stop codons resulted in a 300-fold increase of beta-gal activity compared to controls. A 2-3-fold increase in efficiency was obtained through an elongation of the binding domains. Replacement of the complete 3'end of the COL17A1 gene was shown using a collagen XVII mini-gene construct. The beta-gal assay was used in human keratinocytes to evaluate the influence of a keratinocyte-specific spliceosome background. Reverse transcription polymerase chain reaction and beta-gal activity assay showed functional correction of the stop-codons in cultured human keratinocytes and in an immortalized GABEB cell line harbouring the 4003delTC mutation. These results demonstrate that SMaRT is feasible in a keratinocyte-specific context and therefore may be applied in skin gene therapy.

Partial correction of endogenous DeltaF508 CFTR in human cystic fibrosis airway epithelia by spliceosome-mediated RNA trans-splicing.

Spliceosome-mediated RNA trans-splicing (SMaRT) was investigated as a means for functionally correcting endogenous DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) transcripts using in vitro human cystic fibrosis (CF) polarized airway epithelia and in vivo human CF bronchial xenografts. Recombinant adenovirus (Ad.CFTR-PTM) encoding a pre-therapeutic molecule (PTM) targeted to CFTR intron 9 corrected transepithelial cyclic AMP (cAMP)-sensitive short-circuit current (Isc) in DeltaF508 homozygous epithelia to a level 16% of that observed in normal human bronchial epithelia. Molecular analyses using RT-PCR and western blotting confirmed SMaRT-mediated partial correction of endogenous DeltaF508 messenger RNA (mRNA) transcripts and protein. In an in vivo model of DeltaF508 CF airway epithelia, human CF bronchial xenografts infected with Ad.CFTR-PTM also demonstrated partial correction of CFTR-mediated Cl- permeability at a level 22% of that seen in non-CF xenografts. These results provide functional evidence for SMaRT-mediated repair of mutant endogenous CFTR mRNA in intact polarized CF airway epithelial models.

Messenger RNA repair and restoration of protein function by spliceosome-mediated RNA trans-splicing.

The functional repertoire of the human genome is amplified by the differential assortment of exons. Spliceosome-mediated RNA trans-splicing can mobilize these packets of genetic information to reprogram mRNAs. In principle, this process could repair defective transcripts in loss-of-function genetic disorders in humans. We developed a tractable lacZ repair system to serve as a model for these genetic disorders. Targeted pre-trans-splicing RNA molecules efficiently and specifically repaired mutated lacZ transcripts and restored enzymatic activity in human cells. The development of this model confirms the potential for spliceosome-mediated RNA trans-splicing in genetic repairs and provides a powerful tool for rational design and in vitro evolution of pre-trans-splicing molecules.

Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing.

Most messenger RNA precursors (pre-mRNA) undergo cis-splicing in which introns are excised and the adjoining exons from a single pre-mRNA are ligated together to form mature messenger RNA. This reaction is driven by a complex known as the spliceosome. Spliceosomes can also combine sequences from two independently transcribed pre-mRNAs in a process known as trans-splicing. Spliceosome-mediated RNA trans-splicing (SMaRT) is an emerging technology in which RNA pre-therapeutic molecules (PTMs) are designed to recode a specific pre-mRNA by suppressing cis-splicing while enhancing trans-splicing between the PTM and its pre-mRNA target. This study examined the feasibility of SMaRT as a potential therapy for genetic diseases to correct mutations using cystic fibrosis (CF) as an example. We used several versions of a cystic fibrosis transmembrane conductance regulator (CFTR) mini-gene expressing mutant (deltaF508) pre-mRNA targets and tested this against a number of PTMs capable of binding to the CFTR target intron 9 and trans-splicing in the normal coding sequences for exons 10-24 (containing F508). When 293T cells were cotransfected with both constructs, they produced a trans-spliced mRNA in which normal exon 10-24 replaced mutant exon 10. To test whether SMaRT produced mature CFTR protein, proteins were immunoprecipitated from lysates of cotransfected cells and detected by Western blotting and PKA-phosphorylation. Tryptic phosphopeptide mapping confirmed the identity of CFTR. This proof-of-concept study demonstrates that exon replacement by SMaRT can repair an abnormal pre-mRNA associated with a genetic disease.

Spliceosome-mediated RNA trans-splicing as a tool for gene therapy.

We have developed RNA molecules capable of effecting spliceosome-mediated RNA trans-splicing reactions with a target messenger RNA precursor (pre-mRNA). Targeted trans-splicing was demonstrated in a HeLa nuclear extract, cultured human cells, and H1299 human lung cancer tumors in athymic mice. Trans-splicing between a cancer-associated pre-mRNA encoding the beta-subunit of human chorionic gonadotropin gene 6 and pre-trans-splicing molecule (PTM) RNA was accurate both in vitro and in vivo. Comparison of targeted versus nontargeted trans-splicing revealed a moderate level of specificity, which was improved by the addition of an internal inverted repeat encompassing the PTM splice site. Competition between cis- and trans-splicing demonstrated that cis-splicing can be inhibited by trans-splicing. RNA repair in a splicing model of a nonfunctional lacZ transcript was effected in cells by a PTM, which restored significant beta-galactosidase activity. These observations suggest that spliceosome-mediated RNA trans-splicing may represent a general approach for reprogramming the sequence of targeted transcripts, providing a novel approach to gene therapy.

Circular ribozymes generated in Escherichia coli using group I self-splicing permuted intron-exon sequences.

A circularly permuted self-splicing group I intron from Anabaena was used to generate covalently closed circular trans-acting ribozymes in Escherichia coli. The RNA component of Bacillus subtilis RNaseP and an artificial trans-acting hepatitis delta virus ribozyme were expressed as the exon portion of the permuted intron. RNA isolated from these cells contained circular forms of the ribozymes, indicating that circles were generated from precursors expressed in these cells. Total RNA isolated from cells producing the circular RNA contained ribozyme activity. In contrast, a linear form of the delta virus ribozyme expressed as part of an unprocessed transcript yielded no detectable activity. These data extend previous in vitro and in vivo studies on splicing-mediated RNA circularization by demonstrating the intracellular production of circular ribozymes. These results have implications for the development of systems expressing therapeutic forms of small RNAs such as ribozymes and decoy-type competitors. Circular RNAs generated by splicing are devoid of flanking sequences that could potentially interfere with function. Also, because circular RNAs are not primary substrates for exonucleases, they may have increased in vivo half-lives relative to linear molecules with similar sequences.

A small circular TAR RNA decoy specifically inhibits Tat-activated HIV-1 transcription.

Linear TAR RNA has previously been used as a decoy to inhibit HIV-1 transcription in vitro and HIV-1 replication in vivo. A 48 nucleotide circular RNA containing the stem, bulge and loop of the HIV-1 TAR element was synthesized using the self-splicing activity of a group I permuted intron-exon and was tested for its ability to function as a TAR decoy in vitro. This small circular TAR molecule was exceptionally stable in HeLa nuclear extracts, whereas a similar linear TAR molecule was rapidly degraded. The TAR circle bound specifically to Tfr38, a peptide containing the TAR-binding region of Tat. The ability of Tat to trans-activate transcription from the HIV-1 promoter in vitro was efficiently inhibited by circular TAR RNA but not by TAR circles that contained either bulge or loop mutations. TAR circles did not inhibit transactivation exclusively by binding to Tat since this inhibition was not reversed by adding excess Tat to the transcription reaction. Together, these data suggest that TAR circles act as decoys that inhibit transactivation by binding to Tat and at least one cellular factor. These data also demonstrate the utility of small circular RNA molecules as tools for biochemical studies.

Circularizing ribozymes and decoy-competitors by autocatalytic splicing in vitro and in vivo.

An Anabaena group I intron was circularly permuted at loop 5, loop 6 and loop 8, and tested for self-splicing activity. Precursor RNAs from these constructs spliced efficiently and produced circular exons in vitro. Using group I permuted-intron-exon sequences, circular forms of the HDV ribozyme, the RNA component of RNaseP from B. subtilis, the HIV TAR and a short HIV Rev-binding element were generated and tested for activity and stability. The activity of circular ribozymes is comparable to the linear counterparts with similar core sequences. Circular forms of the TAR and Rev-binding element showed specific binding to Tfr-38 and Rev(35-50) peptide, respectively. To explore the potential for using this methodology to express circular RNA in vivo, circular forms of the HDV ribozyme and RNaseP RNA were produced in E. coli. Analysis of total RNA indicated that the precursor RNA spliced efficiently and accurately to produce circular ribozymes. The activity of in vivo expressed circular ribozymes could be demonstrated indicating that they fold into active conformation. These results suggest that self-splicing group I PIE sequences could prove useful for expressing small stable circular ribozyme/decoy-competitor or antisense RNAs in cells.

Generation of nuclease resistant circular RNA decoys for HIV-Tat and HIV-Rev by autocatalytic splicing.

Circular exon sequences can be generated by splicing permuted intron-exon (PIE) sequences. The Anabaena pre-tRNA group I self-splicing PIE sequence was modified to generate circular forms of the HIV-TAR and the high affinity region of the HIV-RRE (RBE). RNA products containing TAR and the RBE were purified from splicing reactions and demonstrated to be circular. The circular form of these sequences was shown to be resistant to nuclease degradation in cellular extracts. Gel shift assays demonstrate that the circular form of the RBE is specifically bound by a Rev derived peptide. These data suggest that PIE-circularization of RNA may be an effective way to express small stable RNAs designed for therapeutics (eg-decoys).

Generation of nuclease resistant circular RNA decoys for HIV-Tat and HIV-Rev by autocatalytic splicing.

Circular exon sequences can be generated by splicing permuted intron-exon (PIE) sequences. The Anabaena pre-tRNA group I self-splicing PIE sequence was modified to generate circular forms of the HIV-TAR and the high affinity region of the HIV-RRE (RBE). RNA products containing TAR and the RBE were purified from splicing reactions and demonstrated to be circular. The circular form of these sequences was shown to be resistant to nuclease degradation in cellular extracts. Gel shift assays demonstrate that the circular form of the RBE is specifically bound by a Rev derived peptide. These data suggest that PIE-circularization of RNA may be an effective way to express small stable RNAs designed for therapeutics (eg. decoys).

Generation and characterization of circular Bacillus subtilis RNase P RNA; activation by RNase P protein.

A circular form of Bacillus subtilis ribonuclease P RNA (C-P RNA) was generated in vitro by splicing permuted intron-exon (PIE) sequences containing the P RNA sequence. Steady-state cleavage of pre-tRNA(Asp) catalyzed by circular P RNA is slightly faster than the linear form. Furthermore, steady-state turnover catalyzed by circular RNase P RNA is activated by the addition of the Bacillus subtilis protein component of RNase P, to a rate constant equal to the linear holoenzyme under identical conditions. Also, the circles are resistant to nuclease degradation, have less sequence heterogeneity, and may enhance the formation of a unique structure. Therefore, circular forms of RNase P RNA should prove useful for mutagenesis and structural studies.

Identification and characterization of thionucleosides in the total tRNA of cucumber cotyledons.

Total tRNA isolated from cucumber cotyledons grown in the presence of radioactive sulfur was analyzed for the occurrence of thionucleosides. The analysis revealed the presence of at least five thionucleosides which were identified as 5-methylaminomethyl-2-thiouridine (mnm5s2U), 2-methylthio-N6-isopentenyladenosine (ms2i6A), 2-methylthio-N6-hydroxyisopentenyladenosine (ms2io6A), 5-methyl-2-thiouridine (m5s2U) and N-[(9-beta-ribofuranosyl-2- methylthiopurine-2-yl)-carbamoyl]-threonine (ms2t6A). A comparison of relative amounts of these thionucleosides in the total tRNAs of dark-, and light-grown cotyledons shows that the relative amounts of ms2i6A, ms2io6A and ms2t6A remain unchanged whereas mnm5s2U increases with a concomitant decrease in the relative amounts of m5s2U after light treatment of dark-grown cotyledons.

Fractionation and identification of cytoplasmic tRNAS and structural characterization of a phenylalanine and a leucine tRNA from cucumber hypocotyls.

Total cytoplasmic tRNAs from cucumber hypocotyls were fractionated by two-dimensional polyacrylamide gel electrophoresis into about 56 species. Of these, 32 tRNA species were identified by aminoacylation as tRNA specific for 15 amino acids. Furthermore, cytoplasmic tRNA(Phe) and tRNA(Leu(NAA)) were purified to homogeneity by the combination of RPC-5 column chromatography and two-dimensional polyacrylamide gel electrophoresis. The primary structure of tRNA(Leu(NAA)) and the modified nucleotides of tRNA(Phe) have been determined. The sequence of cucumber cytoplasmic tRNA(Leu(NAA)) is identical to that of bean cytoplasmic tRNA(Leu(NAA)) except for three base differences in the variable loop.