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At least 1,009 records · Page 56Linked to original sources
PubMed2002

Comparative analysis of antisense RNA, double-stranded RNA, and delta ribozyme-mediated gene regulation in Toxoplasma gondii.

RNA tools, namely, antisense RNA, double-stranded RNA (dsRNA), and delta ribozyme, were comparatively analyzed for the development of effective RNA-based gene modulators. The gene encoding uracil phosphoribosyltransferase (UPRT) of Toxoplasma gondii was used as a target and a negative selectable marker. Using plasmid transformation and drug selection assays, we obtained T. gondii transformants resistant to 5-fluoro-2'-deoxyuridine (FDUR), the cytotoxic prodrug and substrate of UPRT, when the plasmids expressing dsRNA and active delta ribozyme were used. No resistant transformants were detected when the plasmids carrying the antisense RNA, the inactive delta ribozyme, or the chloramphenicol acetyltransferase (CAT) genes were used. Parasites generated using the plasmids expressing dsRNA and the delta ribozyme become resistant to FDUR with an LD50 of 50 +/- 5 microM and 25 +/- 8 microM, respectively. These values are approximately 25-fold and 12-fold higher than that of the RH parental parasite strain, indicating that UPRT activity of the transformed parasites was drastically inhibited. Using Northern and Southern blot analysis, we demonstrated that dsRNA and the delta ribozyme interrupt the expression of UPRT. These two RNA tools should, thus, be very useful for the study of gene expression.

Animals↗
PubMed2026

DREAMS illuminates spatial DNA and RNA modification landscapes.

DNA and RNA modifications regulate gene expression and RNA processing, but their spatial organization in complex tissues remains elusive. Here we developed DNA RNA Elements Areal Mass Spectrometry (DREAMS), a mass spectrometry imaging platform that spatially maps diverse nucleic acid modifications simultaneously. Applying DREAMS to TET-deficient mouse brains (Tet1Δ/Δ and triple Tet1/2/3Δ/Δ), we uncover TET1's unexpected role in modulating N1-methyladenosine (m1A), a pivotal RNA modification. While DREAMS reveals broad modification landscapes altered across TET knockouts, we identify TET1-mediated changes in m1A that correlate with transcriptome alterations. Our work establishes DREAMS as a transformative tool for spatial epigenomics/epitranscriptomics and suggests that TET enzymes could influence multiple DNA and RNA modifications with potential gatekeeping roles in nucleic acid regulation.

Animals↗
PubMed2001

Characterization of two evolutionarily conserved, alternatively spliced nuclear phosphoproteins, NFAR-1 and -2, that function in mRNA processing and interact with the double-stranded RNA-dependent protein kinase, PKR.

We report here the isolation and characterization of two proteins, NFAR-1 and -2, which were isolated through their ability to interact with the dsRNA-dependent protein kinase, PKR. The NFAR proteins, of 90 and 110 kDa, are derived from a single gene through alternative splicing and are evolutionarily conserved nuclear phosphoproteins that interact with double-stranded RNA. Both NFAR-1 and -2 are phosphorylated by PKR, reciprocally co-immunoprecipitate with PKR, and colocalize with the kinase in a diffuse nuclear pattern within the cell. Transfection studies indicate that the NFARs regulate gene expression at the level of transcription, probably during the processing of pre-mRNAs, an activity that was increased in fibroblasts lacking PKR. Subsequent functional analyses indicated that amino acids important for NFAR's activity were localized to the C terminus of the protein, a region that was found to specifically interact with FUS and SMN, proteins also known as regulators of RNA processing. Accordingly, both NFARs were found to associate with both pre-mRNAs and spliced mRNAs in post-transcriptional studies, similar to the known splicing factor ASF/SF-2. Collectively, our data indicate that the NFARs may facilitate double-stranded RNA-regulated gene expression at the level of post-transcription and possibly contribute to host defense-related mechanisms in the cell.

Alternative Splicing↗
PubMed2001

Differential roles of phosphorylation in the formation of transcriptional active RNA polymerase I.

Regulation of rDNA transcription depends on the formation and dissociation of a functional complex between RNA polymerase I (pol I) and transcription initiation factor Rrn3p. We analyzed whether phosphorylation is involved in this molecular switch. Rrn3p is a phosphoprotein that is predominantly phosphorylated in vivo when it is not bound to pol I. In vitro, Rrn3p is able both to associate with pol I and to enter the transcription cycle in its nonphosphorylated form. By contrast, phosphorylation of pol I is required to form a stable pol I-Rrn3p complex for efficient transcription initiation. Furthermore, association of pol I with Rrn3p correlates with a change in the phosphorylation state of pol I in vivo. We suggest that phosphorylation at specific sites of pol I is a prerequisite for proper transcription initiation and that phosphorylation/dephosphorylation of pol I is one possibility to modulate cellular rDNA transcription activity.

Escherichia coli↗
PubMed2003

tmRNA in Caulobacter crescentus is cell cycle regulated by temporally controlled transcription and RNA degradation.

SsrA, or tmRNA, is a small RNA found in all bacteria that intervenes in selected translation reactions to target the nascent polypeptide for rapid proteolysis. We have found that the abundance of SsrA RNA in Caulobacter crescentus is regulated with respect to the cell cycle. SsrA RNA abundance increases in late G(1) phase, peaks during the G(1)-S transition, and declines in early S phase, in keeping with the reported role for SsrA in the timing of DNA replication initiation. Cell cycle regulation of SsrA RNA is accomplished by a combination of temporally controlled transcription and regulated RNA degradation. Transcription from the ssrA promoter peaks late in G(1), just before the peak in SsrA RNA abundance. SsrA RNA is stable in G(1)-phase cells and late S-phase cells but is degraded with a half-life of 4 to 5 min at the onset of S phase. This degradation is surprising, since SsrA RNA is both highly structured and highly abundant. This is the first observation of a structural RNA that is cell cycle regulated.

Caulobacter crescentus↗
PubMed2003

Methylation of SPT5 regulates its interaction with RNA polymerase II and transcriptional elongation properties.

SPT5 and its binding partner SPT4 function in both positively and negatively regulating transcriptional elongation. The demonstration that SPT5 and RNA polymerase II are targets for phosphorylation by CDK9/cyclin T1 indicates that posttranslational modifications of these factors are important in regulating the elongation process. In this study, we utilized a biochemical approach to demonstrate that SPT5 was specifically associated with the protein arginine methyltransferases PRMT1 and PRMT5 and that SPT5 methylation regulated its interaction with RNA polymerase II. Specific arginine residues in SPT5 that are methylated by these enzymes were identified and demonstrated to be important in regulating its promoter association and subsequent effects on transcriptional elongation. These results suggest that methylation of SPT5 is an important posttranslational modification that is involved in regulating its transcriptional elongation properties in response to viral and cellular factors.

Arginine↗
PubMed1986

A signal regulating mouse histone H4 mRNA levels in a mammalian cell cycle mutant and sequences controlling RNA 3' processing are both contained within the same 80-bp fragment.

Fragments from the 3' end of a mouse histone H4 gene, when introduced into transcription units controlled by the SV40 early promoter, yield correctly processed RNA with histone-specific 3' ends, both in monkey and mouse cell lines. The processed RNA is regulated in parallel with endogenous H4 mRNAs in 21-Tb cells, a temperature-sensitive mouse mastocytoma cell cycle mutant that is specifically blocked in G1 phase at the non-permissive temperature. Mutational analyses of the H4 gene fragment indicate that the minimal sequences for this regulation and for RNA 3' processing are both contained within the same 80 bp. This fragment contains two histone-specific, highly conserved sequence elements that are located at the 3' end of histone mRNA and in the adjacent spacer region, respectively. Our data suggest that the observed cell cycle regulation is achieved either at RNA 3' processing or at some later step involving the conserved 3'-terminal sequence element of mature histone mRNA.

Animals↗
PubMed1992

The RNA polymerase II elongation complex. Factor-dependent transcription elongation involves nascent RNA cleavage.

Regulation of transcription elongation is an important mechanism in controlling eukaryotic gene expression. SII is an RNA polymerase II-binding protein that stimulates transcription elongation and also activates nascent transcript cleavage by RNA polymerase II in elongation complexes in vitro (Reines, D. (1992) J. Biol. Chem. 267, 3795-3800). Here we show that SII-dependent in vitro transcription through an arrest site in a human gene is preceded by nascent transcript cleavage. RNA cleavage appeared to be an obligatory step in the SII activation process. Recombinant SII activated cleavage while a truncated derivative lacking polymerase binding activity did not. Cleavage was not restricted to an elongation complex arrested at this particular site, showing that nascent RNA hydrolysis is a general property of RNA polymerase II elongation complexes. These data support a model whereby SII stimulates elongation via a ribonuclease activity of the elongation complex.

Animals↗
PubMed1998

Developmental regulation of two nuclear RNA binding proteins, p34 and p37, from Trypanosoma brucei.

We have previously reported the purification of two closely related nucleic acid binding proteins, p34 and p37, from Trypanosoma brucei and the cloning and sequencing of the two genes encoding these two proteins. The predicted primary structures of the two proteins are nearly identical with one major and several minor differences. Three sequence motifs have been identified in both proteins: an N-terminal alanine, proline, and lysine rich domain, one and a half internal consensus RNA binding domains, and a C-terminal KKDX repeat region. p34 and p37 bind preferentially to heterogeneous RNA as compared with other nucleic acids. Here, we report the developmental regulation of the expression of these two highly related proteins and their intracellular localization in T. brucei. The results indicate that these two RNA binding proteins are differently regulated through the Trypanosoma brucei life cycle. The steady state level of p34 transcript and protein are highest in the procyclic form. In bloodstream form, however, the p34 message is readily detectable, while the protein is not detectable. The p37 transcript level is nearly as high as that for p34 in procyclic form, while the p37 protein level is low. In bloodstream form p37 protein does correlate with the relative abundance of the steady state mRNA level. The two proteins have been localized to the nucleus by immunofluorescent confocal microscopy and subcellular fractionation.

Amino Acid Sequence↗
PubMed2001

Group A streptococcal growth phase-associated virulence factor regulation by a novel operon (Fas) with homologies to two-component-type regulators requires a small RNA molecule.

A novel growth phase-associated two-component-type regulator, Fas (fibronectin/fibrinogen binding/haemolytic activity/streptokinase regulator), of Streptococcus pyogenes was identified in the M1 genome sequence, based on homologies to the histidine protein kinase (HPK) and response regulator (RR) part of the Staphylococcus aureus Agr and Streptococcus pneumoniae Com quorum-sensing systems. The fas operon, present in all 12 tested M serotypes, was transcribed as polycystronic message (fasBCA) and contained genes encoding two potential HPKs (FasB and FasC) and one RR (FasA). Downstream of fasBCA, we identified a small 300 nucleotide monocistronic transcript, designated fasX, that did not appear to encode true peptide sequences. Measurements of luciferase promoter fusions revealed a growth phase-associated transcription of fasBCA and fasX, with peak activities during the late exponential phase. Insertional mutagenesis disrupting fasBCA and fasA led to a phenotype similar to agr-null mutations in S. aureus, with prolonged expression of extracellular matrix protein-binding adhesins and reduced expression of secreted virulence factors such as streptokinase and streptolysin S. In addition, fasX transcription was dependent on the RR FasA; however, deletion mutagenesis of fasX resulted in a similar phenotype to that of the fasBCA or fasA mutants. Complementation of the fasX deletion mutant, with the fasX gene expressed in trans from a plasmid, restored the wild-type fasBCA regulation pattern. This strongly suggested that fasX, a putative non-translated RNA, is the main effector molecule of the fas regulon. However, using spent culture supernatants from wild-type and fas mutant strains, we were not able to show an influence on the logarithmic growth phase expression of fas and dependent genes. Thus, despite structural and functional similarities between fas and agr, to date the fas operon appears not to be involved in group A streptococcal (GAS) quorum-sensing regulation.

Amino Acid Sequence↗
PubMed2002

RNA silencing: small RNAs as ubiquitous regulators of gene expression.

'RNA silencing' is the suppression of gene expression through nucleotide sequence-specific interactions that are mediated by RNA. Initially identified as an immune system that is targeted against transposons and viruses, RNA silencing is emerging as a fundamental regulatory process that is likely to affect many layers of endogenous gene expression in most, if not all, eukaryotes.

Animals↗
PubMed1999

Identification of the poly(C) binding protein in the complex associated with the 3' untranslated region of erythropoietin messenger RNA.

Hypoxia regulates expression of erythropoietin (EPO), a glycoprotein that stimulates erythrocytosis, at the level of transcription and also possibly at the level of messenger RNA (mRNA) stability. A pyrimidine-rich region within the EPO mRNA 3' untranslated region was implicated in regulation of EPO mRNA stability element and shown to bind protein factors. In the present study we wished to identify the protein factor binding to the pyrimidine-rich sequence in the EPO mRNA stability element. Using mobility shift assays, ultraviolet light cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electroelution of protein factors from the gel slices corresponding to the ribonucleoprotein complexes, we found that two isoforms of a 40 kD poly(C) binding protein (PCBP, also known as alphaCP or hnRNPE), PCBP1, and PCBP2 are present in that complex. In Hep3B or HepG2 cells hypoxia induces neither expression of PCBP nor formation of the ribonucleoprotein complex associated with EPO mRNA that involves PCBP.

3' Untranslated Regions↗
PubMed2003

SRp55 is a regulator of calcitonin/CGRP alternative RNA splicing.

Alternative splicing is an important mechanism for the regulation of gene expression. The mammalian calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA is alternatively spliced in a tissue-specific manner, leading to the production of calcitonin mRNA containing exons 1-4 in thyroid C cells and CGRP mRNA containing exons 1-3, 5, and 6 in neurons. The calcitonin-specific fourth exon contains an exonic splice enhancer (ESE) that binds SRp55. We define the RNA binding site of SRp55 in the ESE and demonstrate that base changes that decrease the level of SRp55 binding decrease the level of calcitonin splicing in vitro and calcitonin mRNA production in vivo. Base changes that increase the affinity of SRp55 for the ESE increase the level of calcitonin splicing in vitro and calcitonin mRNA levels in 293 cells. We also observe that SRp55 levels in different cell types correlate with the levels of calcitonin mRNA produced in these cells. Finally, we show that increasing the level of cellular expression of SRp55 stimulates calcitonin mRNA production in vivo. These observations suggest that SRp55 binding to a suboptimal RNA binding site in the calcitonin/CGRP pre-mRNA ESE is required for calcitonin mRNA production. Differential amounts of SRp55 present in different cell types would then control calcitonin/CGRP alternative splicing.

Alternative Splicing↗
PubMed1990

Apolipoprotein B messenger RNA editing is developmentally regulated and widely expressed in human tissues.

Apolipoprotein B (apoB) messenger RNA (mRNA) undergoes posttranscriptional (C to U) editing, producing tissue-specific forms of apoB from the unedited (apoB100) and edited (apoB48) transcripts. ApoB mRNA editing is developmentally regulated in human fetal small intestine, producing parallel changes in the synthesis and secretion of apoB100 and apoB48. Neither fetal nor adult liver contained detectable levels (less than 1%) of edited apoB mRNA. By contrast, apoB mRNA is incrementally edited during development in several extraintestinal fetal tissues. These tissues demonstrated significant differences, however, in both the extent and developmental profile of apoB mRNA editing. Additionally, while apoB100 synthesis and secretion was demonstrated in these tissues, apoB48 synthesis was confined to the small intestine. Thus, although the molecular mechanism underlying this process remains unclear, the capacity to edit apoB mRNA is widely distributed among both fetal and adult tissues. We speculate that the asynchronous and developmentally modulated induction of apoB mRNA editing may provide a novel mechanism for regulating lipoprotein biogenesis in both the developing fetus and adult human.

Adult↗
PubMed1999

Developmental regulation of spliced leader RNA gene in Leishmania donovani amastigotes is mediated by specific polyadenylation.

Leishmania cycles between the insect vector and its mammalian host undergoing several important changes mediated by the stage-specific expression of a number of genes. Using a genomic differential screening approach, we isolated differentially expressed cosmid clones carrying several copies of the mini-exon gene. We report that the spliced leader (SL) RNA, essential for the maturation of all pre-mRNAs by trans-splicing, is developmentally regulated in Leishmania donovani amastigotes and that this regulation is rapidly induced upon parasite growth under acidic conditions. Stage-specific regulation of the SL RNA is associated with the expression of a larger approximately 170-nucleotide transcript that bears an additional 15-nucleotide sequence at its 3'-end and is polyadenylated in contrast to the mature SL RNA. The poly(A)+ SL RNA represents 12-16% of the total SL transcript synthesized in amastigotes and is 2.5-3-fold more stable than the poly(A)- transcript. The poly(A)+ SL transcript is synthesized specifically from one class of the genomic mini-exon copies. Polyadenylation of the SL RNA may control the levels of the SL mature transcript under amastigote growth and may represent an additional step in the gene regulation process during parasite differentiation.

Animals↗
PubMed2003

Positive and negative regulators for neuronal BC1 RNA transcription by RNA polymerase III are possible members of the RNA polymerase II transcription system.

Neuronal cell-specific BC1 RNA is a unique RNA polymerase III (Pol III) transcript. The transcription is controlled by an activator E2 site and by BCRE, a repressor element, in response to neuronal activity. BC1 RNA is localized to dendritic domains as ribonucleoprotein particles, and it has been suggested to play a functional role in translational regulation of dendritic mRNAs. In the present study, using a luciferase assay in NG108-15 cells, we found that the positive and negative regulators for BC1 RNA transcription can also function in the Pol II transcription system. Our results suggest that the neuronal activity-dependent expression of BC1 RNA by Pol III and a subset of neuronal mRNAs by Pol II may be simultaneously controlled by the E2 site and BCRE, as well as their binding proteins.

Animals↗
PubMed2001

A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA.

The 21-nucleotide small temporal RNA (stRNA) let-7 regulates developmental timing in Caenorhabditis elegans and probably in other bilateral animals. We present in vivo and in vitro evidence that in Drosophila melanogaster a developmentally regulated precursor RNA is cleaved by an RNA interference-like mechanism to produce mature let-7 stRNA. Targeted destruction in cultured human cells of the messenger RNA encoding the enzyme Dicer, which acts in the RNA interference pathway, leads to accumulation of the let-7 precursor. Thus, the RNA interference and stRNA pathways intersect. Both pathways require the RNA-processing enzyme Dicer to produce the active small-RNA component that represses gene expression.

Animals↗
PubMed1999

The role of overlapping U1 and U11 5' splice site sequences in a negative regulator of splicing.

Splicing of Rous sarcoma virus RNA is regulated in part by a cis-acting intronic RNA element called the negative regulator of splicing (NRS). An NRS mutant affecting nt 916-923 disrupts U11 snRNP binding and reduces NRS activity (Gontarek et al., 1993, Genes & Dev 7:1926-1936). However, we observed that a U15' splice site-like sequence, which overlapped the U11 site, was also disrupted by this mutation. To determine whether the U1 or the U11 site was essential for NRS activity, we analyzed twelve additional mutants involving nt 915-926. All mutations that disrupted the potential base pairing between U1 snRNA and the NRS reduced NRS activity, including single point mutations at nt 915, 916, and 919. The point mutation at nt 919 was partially suppressed by a compensatory base change mutation in U1 snRNA. In contrast, a mutation which strengthened the potential base pairing between the U1 site and the NRS increased NRS activity. Surprisingly, mutations that specifically targeted the U115' splice site consensus sequence increased the levels of unspliced RNA, suggesting U11 binding plays an antagonistic role to NRS activity. We propose that U1 snRNP binding to the NRS inhibits splicing and is regulated by U11 snRNP binding to the overlapping sequence. Competition between U1 and U11 snRNPs would result in the appropriate balance of spliced to unspliced RNAs for optimal viral replication. Further, a virus mutated in the U1/U11 region of the NRS was found to have delayed replication.

Avian Sarcoma Viruses↗