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

---

Query: EC:3.1.26.4 (RNase H)
2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The two gonadotropins, LH and FSH, are thought to be synthesized and secreted solely by the anterior pituitary. We present here evidence for expression of the LH beta and common alpha-subunit (C alpha) genes in the rat testis. The LH beta and C alpha-subunit messenger RNAs (mRNAs) were detected by reverse transcriptase-polymerase chain reaction in the rat testis and pituitary with primer pairs producing 247- and 199-base pair complementary DNA (cDNA) fragments, corresponding to nucleotides 154-400 of LH beta and nucleotides 250-448 of C alpha cDNA, respectively. The specificity of the cDNA species generated was verified by Southern hybridization using nested [32P]cDNA or oligonucleotide probes, and identity with the published rat LH beta and C alpha-subunit gene structures was determined by sequencing. The mRNA bands with specific hybridization to complementary RNA (cRNA) probes corresponding to nucleotides 154-368 of the rat LH beta cDNA and nucleotides 250-448 of the rat C alpha cDNA were found in the rat pituitary and testis by Northern hybridization. The major C alpha mRNA had a size of 0.8 kilobases (kb) in the pituitary and testis. The major LH beta transcripts were 0.8 and 2.7 kb in the pituitary and testis, respectively. To further characterize the larger testicular LH beta-subunit transcript, rapid amplification of the 3'-end of cDNA (3'-RACE) was performed using an oligo(deoxythymidine-17) adapter and a specific 5'-primer. Southern hybridization of the 3'-RACE product of rat testicular RNA with a LH beta [32P]cDNA probe had the same size as the 3'-RACE product of pituitary RNA. The pituitary and testicular RNAs were then cut into two segments using oligonucleotide-directed ribonuclease H digestion and subjected to Northern hybridization using a cRNA probe specific to the 5'-end segment. The digested 5'-end segments of the pituitary and testicular mRNAs were 0.4 and 2.3 kb, respectively, indicating that the testicular LH beta mRNA has a 1.9-kb 5'-extension, compared to the cognate pituitary mRNA. This was further verified by Northern hybridization using a cRNA probe corresponding to nucleotides -790 to -10 upstream of the pituitary initiation site of LH beta gene transcription. Specific hybridization of a 2.7-kb mRNA transcript was found in the rat testis, but none in the pituitary. Hence, the 3'-end polyadenalytion site of the LH beta mRNA is the same in rat pituitary and testis, and the different transcript sizes are due to a difference at the 5'-end.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:Novel expression of luteinizing hormone subunit genes in the rat testis. 754 May 43

Two distinct class 1 and class 2 rat liver IGF-I mRNAs contain different 5' leader exons, 1 and 2. RNase protection, primer extension, RACE PCR and ribonuclease H mapping established the complete structure of the 5' end of class 1 and class 2 IGF-I mRNAs. Two major transcription start sites in exon 1 yield class 1 IGF-I mRNAs, including 345 or 245 bases of exon 1. Multiple, clustered transcription start sites in exon 2 yield class 2 IGF-I mRNAs with 84-50 bases of exon 2. Cell-free translation of in vitro transcribed IGF-I mRNAs suggests that class 1 and class 2 mRNAs preferentially initiate translation at distinct AUG codons to result in IGF-I precursors with either 48 residue class 1 pre-peptides or 32 residue class 2 pre-peptides. Some translation initiation also occurs at a downstream AUG common to class 1 and 2 mRNAs to yield IGF-I precursors with a 22 residue pre-peptide. Inclusion of microsomal membranes in translations suggests that the three different pre-peptides each function as co-translationally cleaved signal peptides. However, treatment of processed precursors with endoglycosidase H indicates that co-translational processing of precursors with 22 and 32 residue pre-peptides leads to glycosylation of downstream IGF-I precursor sequences whereas co-translational processing of precursors with 48 residue pre-peptide is not associated with glycosylation.
...
PMID:Multiple transcription start sites in the rat insulin-like growth factor-I gene give rise to IGF-I mRNAs that encode different IGF-I precursors and are processed differently in vitro. 827 98

Indole-3-acetic acid (IAA) promotes ethylene biosynthesis in stems of etiolated pea (Pisum sativum L.) seedlings by rapidly increasing the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase mRNA and by enhancing the activity of the enzyme. Two cDNA clones encoding ACC synthase, Ps-ACS1 and Ps-ACS2, were isolated from a cDNA library prepared from the apical hooks of etiolated pea seedlings that had been treated with 100 microns IAA for 4 h. While studying the expression pattern of IAA-induced ACC synthase mRNA, we observed that the probe for Ps-ACS1 hybridized to two transcripts of 1.6 and 1.9 kb on RNA gel blots. The shorter transcript accumulated before the longer one did, indicating that it is not a degradation product of the latter. Because a similar observation, namely hybridization of one ACC synthase probe to two transcripts, has also been reported in other species, we investigated the relationship between the 1.6- and 1.9-kb transcripts. DNA gel blot analysis using the entire cDNA as probe and RNA gel blot analysis using the 3'-untranslated region as probe indicated that both transcripts are encoded by the same gene. Oligonucleotide-directed RNase H mapping showed that the transcripts differ in the sequence of their 5'-ends. Using 5'-RACE to obtain the DNA sequence of the shorter, transcript, we determined that the 1.6-kb transcript (Ps-ACS1b) begins within the second exon of the 1.9-kb transcript (Ps-ACS1a) and lacks the first 383 bases. Thus, Ps-ACS1b does not encode a full-length ACC synthase protein. Because the Ps-ACS1b sequence is identical to that of Ps-ACS1a, including proper splicing of the second intron, Ps-ACS1b appears to result from the use of an alternative, internal promoter.
...
PMID:A gene encoding 1-aminocyclopropane-1-carboxylate (ACC) synthase produces two transcripts: elucidation of a conserved response. 967 1

The mitochondrial genome of Plasmodium falciparum encodes highly fragmented rRNAs. Twenty small RNAs which are putative rRNA fragments have been found and 15 of them have been identified as corresponding to specific regions of rRNA sequence. To investigate the possible interactions between the fragmented rRNAs in the ribosome, we have mapped the ends of many of the small transcripts using primer extension and RNase protection analysis. Results obtained from these studies revealed that some of the rRNA transcripts were longer than the sequences which encode them. To investigate these size discrepancies, we performed 3' RACE PCR analysis and RNase H mapping. These analyses revealed non-encoded oligo(A) tails on some but not all of these small rRNAs. The approximate length of the oligo(A) tail appears to be transcript-specific, with some rRNAs consistently showing longer oligo(A) tails than others. The oligoadenylation of the rRNAs may provide a buffer zone against 3' exonucleolytic attack, thereby preserving the encoded sequences necessary for secondary structure interactions in the ribosome.
...
PMID:The fragmented mitochondrial ribosomal RNAs of Plasmodium falciparum have short A tails. 1032 33

Polyadenylation is a posttranscriptional modification of RNA occurring in prokaryotes, eukaryotes, and organelles. Long poly(A) tails help export eukaryotic mRNAs and promote mRNA stability and translation, whereas the short bacterial tails facilitate RNA decay. The scarcity of polyadenylated RNAs is one of the obstacles for investigators studying bacterial polyadenylation. The two methods described in this chapter were developed to determine how the poly(A) binding protein Hfq affects the polyadenylation of bacterial RNAs. The first is a 3'-RACE protocol specific to oligoadenylated RNA. This method was designed to rapidly collect a large amount of poly(A) containing 3'-terminal sequences to perform statistical analysis. The second method is an RNA sizing protocol to analyze the polyadenylation status of primary transcripts that were not efficiently detected by 3'-RACE. The latter procedure is based on Northern blot analysis of 3'-RNA fragments generated by RNase H. In the presence of a gene-specific methylated chimeric RNA-DNA oligonucleotide, the enzyme is directed to a unique cleavage site. The 3'-RNA fragments, differing by just one nucleotide at their 3'-ends, are then separated in polyacrylamide gels.
...
PMID:The role of RNA chaperone Hfq in poly(A) metabolism methods to determine positions, abundance, and lengths of short oligo(A) tails. 1916 43

Previous studies in our laboratory showed that the RNA debranching enzyme (DBR1) is not required for early steps in HIV cDNA formation but is necessary for synthesis of intermediate and late cDNA products. To further characterize this effect, we evaluated the topology of the 5' end of the HIV-1 RNA genome during early infection with and without inhibition of DBR1 synthesis. Cells were transfected with DBR1 short hairpin RNA (shRNA) followed 48 h later by infection with an HIV-1-derived vector containing an RNase H-deficient reverse transcriptase (RT). RNA was isolated at several times postinfection and treated with various RNA-modifying enzymes prior to rapid amplification of 5' cDNA ends (5' RACE) for HIV-1 RNA and quantitative reverse transcriptase PCR (qRT-PCR). In infected cells, DBR1 knockdown inhibited detection of free HIV-1 RNA 5' ends at all time points. The difference in detection of free HIV-1 RNA 5' ends in infected DBR1 knockdown versus control cells was eliminated by in vitro incubation of infected cell RNAs with yeast or human DBR1 enzyme prior to 5' RACE and qRT-PCR. This was dependent on the 2'-5' phosphatase activity of DBR1, since it did not occur when we used the catalytically inactive DBR1(N85A) mutant. Finally, HIV-1 RNA from infected DBR1 knockdown cells was resistant to RNase R that degrades linear RNAs but not RNAs in circular or lariat-like conformations. These results provide evidence for formation of a lariat-like structure involving the 5' end of HIV-1 RNA during an early step in infection and the involvement of DBR1 in resolving it.IMPORTANCE Our findings support a new view of the early steps in HIV genome replication. We show that the HIV genomic RNA is rapidly decapped and forms a lariat-like structure after entering a cell. The lariat-like structure is subsequently resolved by the cellular enzyme DBR1, leaving a 5' phosphate. This pathway is similar to the formation and resolution of pre-mRNA intron lariats and therefore suggests that similar mechanisms may be used by HIV. Our work therefore opens a new area of investigation in HIV replication and may ultimately uncover new targets for inhibiting HIV replication and for preventing the development of AIDS.
...
PMID:Conformational Changes in the 5' End of the HIV-1 Genome Dependent on the Debranching Enzyme DBR1 during Early Stages of Infection. 2893 90

Alternative polyadenylation is an essential RNA processing event that contributes significantly to regulation of transcriptome diversity and functional dynamics in both animals and plants. Here we review newly developed next generation sequencing methods for genome-wide profiling of alternative polyadenylation (APA) sites, bioinformatics pipelines for data processing and both wet and dry laboratory approaches for APA validation. The library construction methods LITE-Seq (Low-Input 3'-Terminal sequencing) and PAC-seq (PolyA Click sequencing) tag polyA⁺ cDNA, while BAT-seq (BArcoded, three-prime specific sequencing) and PAPERCLIP (Poly(A) binding Protein-mediated mRNA 3'End Retrieval by CrossLinking ImmunoPrecipitation) enrich polyA⁺ RNA. Interestingly, only WTTS-seq (Whole Transcriptome Termini Site sequencing) targets both polyA⁺ RNA and polyA⁺ cDNA. Varieties of bioinformatics pipelines are well established to pursue read quality control, mapping, clustering, characterization and pathway analysis. The RHAPA (RNase H alternative polyadenylation assay) and 3'RACE-seq (3' rapid amplification of cDNA end sequencing) methods directly validate APA sites, while WTSS-seq (whole transcriptome start site sequencing), RNA-seq (RNA sequencing) and public APA databases can serve as indirect validation methods. We hope that these tools, pipelines and resources trigger huge waves of interest in the research community to investigate APA events underlying physiological, pathological and psychological changes and thus understand the information transfer events from genome to phenome relevant to economically important traits in both animals and plants.
...
PMID:Alternative polyadenylation analysis in animals and plants: newly developed strategies for profiling, processing and validation. 3041 85

High-throughput sequencing of the products of 5' RNA ligase-mediated rapid amplification of cDNA ends (5' RLM-RACE) reactions (RACE-SEQ) enables the mapping and digital enumeration of expected and novel 5' ends in RNA molecules. The resulting data are essential in documenting the mechanism of action and precision of endonucleolytically active, RNA-targeting drugs such as RNase H-active antisense or small interfering RNA. When applied to error-prone replication systems such as RNA viruses or in vitro RNA replicon systems, the method can additionally report the relative susceptibility of known and unknown polymorphisms to a prospective sequence-specific drug, making it a powerful tool in patient selection and stratification as well as resistance prediction.We describe the preparation of sequencing libraries for ultra-high depth 5' RLM-RACE analysis on two popular second-generation high-throughput sequencing platforms (Illumina, Ion Torrent) and supply a detailed bioinformatics analysis pipeline for target site activity definition and enumeration. We further illustrate how the pipeline can be simply modified to generate polymorphism-specific drug susceptibility data from in vitro replicon experiments (RACE-SEQ-MM), in a patient-free manner, to cover both known and unknown target site variants in the population.
...
PMID:RACE-SEQ and Population-Wide Polymorphism Susceptibility Testing for Endonucleolytically Active, RNA-Targeting Therapeutics. 3141 Aug 4

---