Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The expression of the major protein kinase C substrate MARCKS (myristoylated alanine-rich C kinase substrate) is controlled by the stability of its mRNA. While the MARCKS mRNA is long living in quiescent fibroblasts (t1/2 = 14 h), its half-life time is drastically reduced (t1/2 = 2 h) in cells treated with phorbol esters to activate protein kinase C (PKC) or treated with growth factors. In a first step to study the underlying mechanism we identified both a cis-element on the MARCKS mRNA and the corresponding trans-acting factors. Fusing the complete 3'-UTR or specific regions of the 3'-UTR of the MARCKS gene to a luciferase reporter gene caused a drastic decrease in luciferase expression to as low as 5-10% of controls. This down-regulation was a result of destabilization of the chimeric transcript as shown by RNA run-off and Northern blot-assays. By RNase/EMSA and UV-cross-linking experiments, we identified a stretch of 52 nucleotides [(CUUU)11(U)8] in the 3'-UTR of the MARCKS mRNA specifically recognized by two RNA-binding proteins, HuD and HuR. These trans-acting factors are members of the ELAV gene family and bind the MARCKS CU-rich sequence with high affinity. Overexpression of HuD and HuR in murine fibroblasts caused a striking stabilization of the endogenous MARCKS mRNA even under conditions when the MARCKS mRNA is normally actively degraded, i.e. after treating cells with phorbol ester. These data imply, that the identified CU-rich cis-element of the MARCKS 3'-UTR is involved in conferring instability to mRNAs and that members of the ELAV gene family oppose this effect. Based on its structural and functional properties, the (CUUU)11(U)8 sequence described here can be grouped into class III of AU-rich elements.
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PMID:The 3'-UTR of the mRNA coding for the major protein kinase C substrate MARCKS contains a novel CU-rich element interacting with the mRNA stabilizing factors HuD and HuR. 1260 86

The RING-finger protein SNURF/RNF4, a modulator of both steroid receptor dependent and basal transcription, is expressed at very high levels in testis and at much lower levels in several other tissues. In somatic tissues, the RNF4 gene is expressed as a 3-kb transcript while an additional shorter sized transcript (1.6 kb) was found in mouse testis. In murine germ cells, RNF4 protein expression is strongly modulated during progression of spermatogonia to spermatids, with a peak in spermatocytes. The expression of 3-kb transcript correlated with protein levels in the different germ cell populations. Conversely, the 1.6-kb transcript was abundantly and specifically expressed in spermatids, in which RNF4 protein was detected at very low levels. We have then examined possible mechanisms underlying this discrepancy. Primer extension and RNase protection analyses demonstrated that the 1.6- and 3.0-kb transcripts originate from the same promoter, encode for the same protein and differ in the 3' UTR. In vitro assays showed that protein degradation is not involved in the regulation of RNF4 protein level. Finally, polysome analysis revealed that only a slight fraction of the testis-specific transcript is engaged in translation, thus providing a feasible mechanism for the quantitative differences of RNF4 mRNA and protein levels. Present results demonstrate that RNF4 short transcript is poorly translated suggesting that this mechanism could be essential for normal spermatogenesis.
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PMID:Translational regulation of a novel testis-specific RNF4 transcript. 1287 92

The process of creating chimeric mRNA transcripts derived from separately transcribed genes via known spliceosome mechanisms is termed trans-splicing, and has been primarily described in lower eukaryotes. Isolation of cDNA clones containing sequences from distinct genes has raised the possibility of trans-splicing across distinct genes in mammalian systems; however, the possibility of cloning artifacts or splicing via nonspliceosome mechanisms has been difficult to rule out. In most cases, the absence of corresponding genomic clones has limited assessment of splice donor and acceptance sites and associated intronic elements that would be expected to participate in spliceosome-based reactions. We have previously reported a cDNA clone encoding the rat Leukocyte Common Antigen-Related (LAR) tyrosine phosphatase receptor that contains an alternative 3' UTR. In the present study Northern, RT-PCR and RNase protection assays verified the existence of developmentally regulated 3' UTR alternative splicing of LAR transcripts in vivo. FISH and radiation hybrid mapping demonstrated that loci encoding LAR and its alternative 3' UTR are present on distinct chromosomes, raising the possibility that alternatively spliced transcripts resulted from trans-splicing. Exon/intron analysis of corresponding genomic clones revealed nonconsensus splice junctions along with elements known to promote both cis- and trans-splicing. Verification in a mammalian in vivo system of chimeric transcripts derived from distinct genes along with identification of atypical nonconsensus-associated genomic elements points to the novel possibilities of atypical spliceosome-based trans-splicing or nonconventional, nonspliceosome-based mechanisms leading to chimeric transcripts.
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PMID:A candidate chimeric mammalian mRNA transcript is derived from distinct chromosomes and is associated with nonconsensus splice junction motifs. 1294 Nov 58

Using modified representational difference analysis, a DNA fragment (GC3) was isolated as a difference between a breast cancer and a normal cell line from the same patient. GC3 proved to be a fragment of intron 7 of the ELF3 gene, an ets family transcription factor, amplified in the breast cancer cell line. Using genomic walking technology, a new Alu (Alu(kwd)) was found downstream of GC3 in an antisense position between nt 8762 and nt 8763 within intron 8 of the ELF3 gene. This ELF3 intron fragment(GC3) was expressed in human breast cancer cell lines and four of six breast cancer tissues, but not in matched normal cell lines and tissues. Similarly, Alu(kwd) was also found in the same breast cancer cell lines and five of eight other breast cancer tissues, but not in matched normal cell lines and tissue. This was confirmed by RNase and DNase digestion analysis. Moreover, GC3 and Alu(kwd) were detected in both the nuclear and cytoplasmic RNA fractions of breast cancer cell lines. The finding of cytoplasmic intron retention was verified with northern blotting and the 5' and 3' rapid amplification cDNA ends procedure (5' and 3'RACE) to search for cDNA sequences in RNA from these cancer cell lines. Partially unspliced ELF3 mRNA and fully spliced ELF3 mRNA was found in the same breast cancer cell line. Partially unspliced ELF3 mRNA contained introns 4-7 without any nucleotide mutation at intron/exon splice junction borders. Fully spliced 1959 bp ELF3 mRNA showed a different 5'UTR from the published ELF3 mRNA, and was predicted to encode a 371 amino acid protein sharing 98% homology with the ELF3 protein sequence. This is the first report of intron retention of ELF3 as well as the pathological appearance of both spliced and unspliced cytoplasmic ELF3 mRNA in human breast cancer cells.
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PMID:Partially unspliced and fully spliced ELF3 mRNA, including a new Alu element in human breast cancer. 1499 48

Our previous studies revealed a splicing variant (lacking a 42 base pair segment) within the 5'-UTR of the ERCC1 gene, a critical component of the nucleotide excision repair (NER) pathway that plays an important role in the development of chemoresistance in platinum-based anticancer therapy. This 42-bp segment seems to possess a regulatory function in ERCC1 expression and representing the level of clinical response to platinum-treatment in ovarian cancer patients. To confirm the existence of the 42-bp deletion and to investigate the 42-bp function, we performed several experiments and assays. Northern blot analysis and RNase protection assay provide evidence that the 42-bp deletion occurs at RNA level of ERCC1 5'-UTR in both ovarian cancer cell lines and ovarian cancer tissues. Luciferase assay suggests that this gene fragment possesses a regulatory function as an enhancer of ERCC1 gene expression in ovarian cancer cells. In Electrophoretic Mobility Shift Assay (EMSA), a shift band present in the ovarian cancer cell line extracts is consistent with the presence of an intracellular protein that recognizes this specific 42-bp sequence. Further, specific EMSA results with 42-bp probe mutated at the site of RFX-1 indicate different putative-DNA binding proteins, rather than RFX-1. We conclude that the 42-bp sequence within the 5'-UTR influences the expression of ERCC1 and hence can influence response to cisplatin in ovarian cancer therapy.
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PMID:Confirmation of 42-bp deletion within the ERCC1 5' UTR. 1537 62

A ribonuclease protection assay (RPA) was developed for the direct detection and quantitation of HCV RNA in infected patients' sera or plasma using HCV [(32)P]RNA from the conserved 5'-untranslated region (5'-UTR) as a probe. We were able to directly detect the presence of HCV RNA by RPA in several infected patients' samples. The viremic status of HCV infected patients with indeterminate recombinant immunoblot assay (RIBA II) was also determined by this assay. Polymerase chain reaction (PCR) was also performed on all these samples and were found to be positive with a concordance of 100% between the results of PCR and RPA. The RPA was able to detect approximately 1 pg of HCV RNA. A limited sequence heterogeneity among HCV isolates was also observed by this assay, suggesting that this may be a faster method of detecting heterogeneous HCV sequences in patients' samples. This simple and specific method could be used to quantitate HCV RNA in order to better determine viremia and follow the course of HCV infection especially when RIBA II results are indeterminate.
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PMID:A ribonuclease protection assay for the direct detection and quantitation of hepatitis C virus RNA. 1556 37

Darkness rapidly induces a decline in the stability and translation of the pea Ferredoxin-1 (Fed-1) mRNA in transgenic tobacco. Direct half-life measurement showed that mutation of the (CAUU)4 stabilizes Fed-1 mRNA in the dark. (CAUU)1, a feature more common in plant 5' UTRs than (CAUU)4, confers slight light-responsive mRNA accumulation. At least three but less than 11 CAUU repeats near the 5' end of the 5' UTR are required for full light-responsive accumulation. Furthermore, 26 nt of the 5' UTR, including the (CAUU)4 repeat, is sufficient to confer a significant approximately 2.5-fold increase in light-regulated mRNA accumulation when fused to the 5' end of a heterologous plant mRNA. A mutation of the (CAUU)4 repeat that compromises light-regulated mRNA stability changes in vitro the accessibility of the region to ribonuclease V1 and ribonuclease A suggesting the geometry formed by the repeat may be important for instability. Finally, dark-induced Fed-1 mRNA instability occurs even when most of the mRNA is retained on polyribosomes, and thus is likely an independent event regulated by darkness.
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PMID:The Fed-1 (CAUU)4 element is a 5' UTR dark-responsive mRNA instability element that functions independently of dark-induced polyribosome dissociation. 1580 13

The serine/threonine kinase pim-1 mRNA contains a long and G/C rich 5'-untranslated region (5'-UTR). Previous work suggested that the pim-1 5'-UTR harbors an internal ribosomal entry site (IRES) allowing for internal initiation of translation. However, several previously reported eukaryotic IRES elements actually contain cryptic promoter activity. To test whether an IRES or a cryptic promoter is present in the pim-1 5'-UTR, the 5'-UTR was re-examined using stringent test procedures. Our results show the presence of strong promoter activity in the DNA sequence corresponding to the pim-1 5'-UTR. Both promoterless dicistronic test and northern blot analysis show transcripts being derived from the cryptic promoter in the pim-1 5'-UTR sequence. This cryptic promoter is active in all cell types tested, including Cos-7, NIH3T3, HEK293, Jurkat and K562 cells. When a dicistronic mRNA containing the pim-1 5'-UTR was translated in vitro or in vivo, no IRES activity could be detected. However, the control IRESs from both human rhinovirus and encephalomyocarditis virus exhibited strong IRES activities. In addition, both the RNase protection assay and the 5'-RACE assay detected endogenous pim-1 transcripts with shorter 5'-UTRs. Our data strongly suggest that the IRES activity reported earlier for the pim-1 5'-UTR sequence is due to cryptic promoter activity.
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PMID:Cryptic promoter activity in the DNA sequence corresponding to the pim-1 5'-UTR. 1584 87

The poly(A)-limiting element (PLE) is a conserved sequence originally found in the 3' UTR of Xenopus albumin mRNA whose presence restricts the length of the poly(A) tail on both pre-mRNA and fully processed mRNA to <20 nt. Results presented in this study show that the PLE also increases the cytoplasmic level of reporter beta-globin mRNA. Transcription run-on shows this increase was not due to increased reporter gene transcription, and experiments with tetracycline repressor-controlled reporter mRNA showed the PLE does not alter the rate of mRNA decay. Both RT-PCR and RNase protection assay showed the PLE caused a 50% increase in the 3' processing of reporter beta-globin mRNA in vivo. This was confirmed in vitro, where PLE-containing RNA was cleaved in HeLa nuclear extract at a rate 80% faster than a control RNA bearing an inactive element. These results indicate that the PLE regulates the length of the poly(A) tail and the efficiency of 3' processing. In addition, they show that PLE-containing mRNA with a <20-nt poly(A) tail is as stable as mRNA with a 100- to 200-nt poly(A) tail.
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PMID:The poly(A)-limiting element enhances mRNA accumulation by increasing the efficiency of pre-mRNA 3' processing. 1587 82

The type I insulin-like growth factor receptor (IGF-IR) is an integral component in the control of cell proliferation, differentiation and apoptosis. The IGF-IR mRNA contains an extraordinarily long (1038 nt) 5'-untranslated region (5'-UTR), and we have characterized a diverse series of proteins interacting with this RNA sequence which may provide for intricate regulation of IGF-IR gene expression at the translational level. Here, we report the purification and identification of one of these IGF-IR 5'-UTR-binding proteins as HuR, using a novel RNA crosslinking/RNase elution strategy. Because HuR has been predominantly characterized as a 3'-UTR-binding protein, enhancing mRNA stability and generally increasing gene expression, we sought to determine whether HuR might serve a different function in the context of its binding the IGF-IR 5'-UTR. We found that HuR consistently repressed translation initiation through the IGF-IR 5'-UTR. The inhibition of translation by HuR was concentration dependent, and could be reversed in trans by addition of a fragment of the IGF-IR 5'-UTR containing the HuR binding sites as a specific competitor, or abrogated by deletion of the third RNA recognition motif of HuR. We determined that HuR repressed translation initiation through the IGF-IR 5'-UTR in cells as well, and that siRNA knockdown of HuR markedly increased IGF-IR protein levels. Interestingly, we also found that HuR potently inhibited IGF-IR translation mediated through internal ribosome entry. Kinetic assays were performed to investigate the mechanism of translation repression by HuR and the dynamic interplay between HuR and the translation apparatus. We found that HuR, occupying a cap-distal position, significantly delayed translation initiation mediated by cap-dependent scanning, but was eventually displaced from its binding site, directly or indirectly, as a consequence of ribosomal scanning. However, HuR perpetually blocked the activity of the IGF-IR IRES, apparently arresting the IRES-associated translation pre-initiation complex in an inactive state. This function of HuR as a 5'-UTR-binding protein and dual-purpose translation repressor may be critical for the precise regulation of IGF-IR expression essential to normal cellular homeostasis.
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PMID:The ELAV RNA-stability factor HuR binds the 5'-untranslated region of the human IGF-IR transcript and differentially represses cap-dependent and IRES-mediated translation. 1591 70


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