Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.26.4 (RNase H)
 2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Stable association of U2 snRNP with the branchpoint sequence of mammalian pre-mRNAs requires binding of a non-snRNP protein to the polypyrimidine tract. In order to determine how U2 snRNP contacts this protein, we have used an RNA containing the consensus 5' and the (Py)n-AG 3' splice sites but lacking the branchpoint sequence so as to prevent direct U2 snRNA base pairing to the branchpoint. Different approaches including electrophoretic separation of RNP complexes formed in nuclear extracts, RNase T1 protection immunoprecipitation assays with antibodies against snRNPs and UV cross-linking experiments coupled to immunoprecipitations allowed us to demonstrate that at least three splicing factors contact this RNA at 0 degree C without ATP. As expected, U1 snRNP interacts with the region comprising the 5' splice site. A protein of approximately 65,000 molecular weight recognizes the RNA specifically at the 5' boundary of the polypyrimidine tract. It could be either the U2 auxiliary factor (U2AF) (Zamore and Green (1989) PNAS 86, 9243-9247), the polypyrimidine tract binding protein (pPTB) (Garcia-Blanco et al. (1989) Genes and Dev. 3, 1874-1886) or a mixture of both. U2 snRNP also contacts the RNA in a way depending on p65 binding, thereby further arguing that the latter may correspond to the previously characterized U2AF and pPTB. Cleavage of U2 snRNA sequence by a complementary oligonucleotide and RNase H led us to conclude that the 5' terminus of U2 snRNA is required to ensure the contact between U2 snRNP and p65 bound to the RNA. More importantly, this conclusion can be extended to authentic pre-mRNAs. When we have used a human beta-globin pre-mRNA instead of the above artificial substrate, RNA bound p65 became precipitable by anti-(U2) RNP and anti-Sm antibodies except when the 5' end of U2 snRNA was selectively cleaved.
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PMID:The 5' end domain of U2 snRNA is required to establish the interaction of U2 snRNP with U2 auxiliary factor(s) during mammalian spliceosome assembly. 185 Jan 27

A series of antisense pentadecamers complementary to a variety of target sequences between the cap and AUG initiation codon regions of c-myc mRNA was synthesized and used to treat human promyelocytic leukemia HL-60 cells. The sensitivity of the cap-region sequences to antisense inhibition of c-myc p65 expression was two to three times that of the original initiation codon antisense sequence. The other target sequences downstream of the cap and up to the AUG initiation codon were comparable to the initiation codon sequence, except that the first splice junction was slightly more sensitive. At the primary initiation codon target, a dodecamer was about half as effective as the original pentadecamer, whereas an octadecamer was about twice as effective. The observation of variation in antisense efficacy as a function of target location in c-myc mRNA may represent a combination of the effects of hybrid arrest, RNase H attack, and interference in RNA processing. Alternatively, the most sensitive targets might be those that are the most exposed in the secondary and tertiary structure of c-myc mRNA.
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PMID:Walking along human c-myc mRNA with antisense oligodeoxynucleotides: maximum efficacy at the 5' cap region. 199 45

Oligonucleotide N3'->P5' Phosphoramidates (PN) may confer advantages over unmodified phosphodiester compounds for therapeutic applications (1). Previous in vitro data demonstrated that PN Oligodeoxynucleotides (ODNs) possess several advantageous features, including RNase H-independence, an improved resistance to nuclease degradation, decreased protein binding, and high affinity sequence-specific binding to complementary RNAs (1, 2). Consequently, we undertook a study to investigate the effects of PN antisense (AS) oligos targeted against the p65 subunit of the Nuclear Factor Kappa beta (NF-kappaB) transcription factor in vivo, in mice. The ability of the antisense molecules to inhibit IL-6 elevation induced by lipopolysaccharide (LPS) in mice, was studied. A 16 mer uniformly modified PN and a chimeric phosphoramidate-phosphodiester oligodeoxynucleotide complementary to the region surrounding the starting codon, (PN-PO-PN) of the NK-kappaB p65 subunit mRNA, both caused a sequence specific reduction of the serum IL-6 level in mice. A scrambled oligodeoxynucleotide showed much lower IL-6 inhibition in mice. These results show that the p65 PN-AS can modulate expression of IL-6 in mice without uptake enhancers and therefore may be a useful prototype for RNAse-H independent therapeutic agents.
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PMID:Inhibition of IL-6 in mice by anti-NF-kappaB oligodeoxyribonucleotide N3'-->oligodeoxyribonnucleotide N3' --> P5' phosphoramidates. 1056 71

Isis 3521 and G3139 are 20- and 18-mer phosphorothioate oligonucleotides, respectively, targeted to the protein kinase C (PKC)-alpha and bcl-2 mRNAs. Treatment of T24 bladder and PC3 prostate carcinoma cells with full-length and 3'-truncation mutants of Isis 3521 causes down-regulation of PKC-alpha protein and mRNA. However, at the level of a 15-mer and shorter, down-regulation of mRNA expression is no longer observed. Further, no diminution in cellular viability, as measured by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide assay, in response to increasing concentrations of paclitaxel, can be observed for these shorter oligomers. These observations not only indicate that PKC-alpha protein expression can be down-regulated by both RNase H-dependent and -independent mechanisms but also that down-regulation of PKC-alpha is insufficient by itself to "chemosensitize" cells. G3139, which down-regulates bcl-2 protein and mRNA expression, also down-regulates PKC-alpha protein and mRNA expression but not that of PKC-betaI, -epsilon, or -zeta. However, the down-regulation of PKC-alpha and bcl-2 are not linked. When the carrier Eufectin 5 is employed, only bcl-2 is down-regulated in both T24 and PC3 cells at 50 nM oligonucleotide concentration. At 100 nM, both bcl-2 and PKC-alpha expression are down-regulated, and only at this concentration can "chemosensitization" to paclitaxel and carboplatin be observed. In contrast, the down-regulation of bcl-2 seems to be linked with that of RelA (p65). However, this too is also not sufficient for chemosensitization, even though it leads to the loss of expression of genes under the putative control of nuclear factor-kappaB and to detachment of the cells from plastic surfaces. These results underscore the complexity of the intracellular requirements for the initiation of chemosensitization to anti-neoplastic agents.
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PMID:Inhibition of potentially anti-apoptotic proteins by antisense protein kinase C-alpha (Isis 3521) and antisense bcl-2 (G3139) phosphorothioate oligodeoxynucleotides: relationship to the decreased viability of T24 bladder and PC3 prostate cancer cells. 1172 37