Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Enhancer RNAs”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 613 records · Page 34Linked to original sources
PubMed1990

Isolation of cDNA clones for the interferon-induced 67,000-dalton protein: direct induction of a family of mRNAs by human interferon-alpha and interferon-gamma.

A partial cDNA clone for the interferon (IFN)-induced 67,000-dalton (67K) protein was isolated by immunological screening and used as a probe to study the expression of mRNAs encoding this protein. Northern blot analyses of RNA from IFN-treated GM2767 cells revealed the presence of two major 67K-specific RNA species, 2.7 and 4.3 kb in length, and two minor RNA species, 5.7 and 7.2 kb long. All of these 67K-specific RNAs were polyadenylated. Multiple 67K-specific mRNAs were observed to be induced in several cell lines. IFN-gamma was more effective at inducing these mRNAs than was IFN-alpha. In IFN-alpha-treated GM2767 cells, the 67K-specific mRNAs were detectable 6 h following IFN treatment, but not 12, 18, or 24 h following treatment. In IFN-gamma-treated cells, these mRNAs were detectable 6 h after treatment and continued to be present 24 h after treatment. The induction of the 67K-specific mRNAs in GM2767 cells did not require protein synthesis as the RNAs were induced by IFN-alpha or IFN-gamma in the presence of cycloheximide (CHX, 50 micrograms/ml). Treatment of cells with the combination of CHX and IFN-alpha mediated an enhanced accumulation of the 67K-specific mRNAs, suggesting that ongoing protein synthesis may downregulate the induction or accumulation of the IFN-alpha-induced 67K-specific mRNAs. Western blot analysis employing a monoclonal antibody to the 67K protein revealed that several distinctly sized but immunologically related proteins were induced in IFN-treated cells.

Cell Line↗
PubMed1984

Physical properties of the E. coli 4.5S RNA: first results suggest a hairpin helix of unusual thermal stability.

Hyperchromicity measurements and quasi-elastic laser light scattering (QELS) have been used to assess the solution structure of the metabolically stable E. coli 4.5S RNA. Results from thermal denaturation measurements revealed the 4.5S species to be markedly more stable than most other RNAs characterized thus far. Optical Tm's range from 79 degrees to 88 degrees with transitions approximately 25 degrees C wide. The Tm values show little dependence on ionic strength, but stability is enhanced considerably by Mg+2. In the QELS experiments the diffusion coefficient does not decrease until T greater than 70 degrees C. Neither the diffusive melting nor the diffusion coefficient at infinite dilution (D0(20,w)) show dependencies on ionic strength but both are influenced by Mg+2. The diffusion behavior is in agreement with that predicted for a rigid cylindrical molecule 125 to 160 A long and 37 to 26 A in diameter. Taken together these results are consistent with the more stable hypothetical secondary structures that can be formed, in which 70-75% of the 114 bases are paired to form a single extended hairpin helix.

Base Sequence↗
PubMed1996

Accumulation of alfalfa mosaic virus RNAs 1 and 2 requires the encoded proteins in cis.

RNAs 1 and 2 of the tripartite genome of alfalfa mosaic virus (A1MV) encode the replicase proteins P1 and P2, respectively. P1 expressed in transgenic plants (P1 plants) can be used in trans to support replication of A1MV RNAs 2 and 3, and P2 expressed in transgenic plants (P2 plants) can be used in trans to support replication of A1MV RNAs 1 and 3. Wild-type RNA 1 was able to coreplicate with RNAs 2 and 3 in P1 plants, but this ability was abolished by frameshifts or deletions in the P1 gene of RNA 1. Similarly, wild-type RNA 2 coreplicated with RNAs 1 and 3 in P2 plants, but frameshifts or deletions in the P2 gene of RNA 2 interfered with this replication. Apparently, the P1 and P2 genes are required in cis for the accumulation of RNAs 1 and 2, respectively. Point mutations in the GDD motif of the P2 gene in RNA 2 interfered with accumulation of RNA 2 in P2 plants, indicating that replication of RNA 2 is linked to its translation into a functional protein. Plants transformed with both the P1 and P2 genes (P12 plants) accumulate replicase activity that is able to replicate RNA 3 in trans. An analysis of the time course of the accumulation of RNAs 1, 2, and 3 in protoplasts of P12 plants supported the conclusion that translation and replication are tightly coupled for A1MV RNAs 1 and 2 but not for RNA 3.

Alfalfa mosaic virus↗
PubMed1999

Two-helper RNA system for production of recombinant Semliki forest virus particles.

Alphavirus expression systems based on suicidal virus particles carrying recombinant replicons have proven to be a very efficient way to deliver genes for heterologous protein expression. However, present strategies for production of such particles have biosafety limitations due to the generation, by RNA recombination, of replication-proficient viruses (RPVs). Here we describe a new packaging system for Semliki Forest virus (SFV) based on a the use of a two-helper system in which the capsid and spike proteins of the C-p62-6K-E1 polyprotein are expressed from two independent RNA molecules. The capsid gene contains a translational enhancer and therefore that sequence was also engineered in front of the spike sequence p62-6K-E1. A sequence coding for the foot-and-mouth disease virus 2A autoprotease was inserted in frame between the capsid translational enhancer and the spike genes. This allows production of the spike proteins at high levels with cotranslational removal of the enhancer sequence and normal biosynthesis of the spike complex. The autoprotease activity of the capsid protein was abolished by mutation, further increasing the biosafety of the system. Cotransfection of cells with both helper RNAs and an SFV vector replicon carrying the LacZ gene led to production of recombinant particles with titers of up to 8 x 10(8) particles per 10(6) cells. Extensive analysis failed to demonstrate the presence of any RPVs, emphasizing the high biosafety of the system based on two-helper RNAs.

Amino Acid Sequence↗
PubMed2001

Genetic analysis of the pestivirus nonstructural coding region: defects in the NS5A unit can be complemented in trans.

The functional analysis of molecular determinants which control the replication of pestiviruses was considerably facilitated by the finding that subgenomic forms of the positive-strand RNA genome of BVDV (bovine viral diarrhea virus) are capable of autonomous replication in transfected host cells. The prototype replicon, BVDV DI9c, consists of the genomic 5' and 3' untranslated regions and a truncated open reading frame (ORF) encoding mainly the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To gain insight into which of these proteins are essential for viral replication and whether they act in cis or in trans, we introduced a large spectrum of in-frame mutations into the DI9c ORF. Tests of the mutant RNAs in terms of their replication capacity and their ability to support translation and cleavage of the nonstructural polyprotein, and whether defects could be rescued in trans, yielded the following results. (i) RNA replication was found to be dependent on the expression of each of the DI9c-encoded mature proteins NS3 to NS5B (and the known associated enzymatic activities). In the same context, a finely balanced molar ratio of the diverse proteolytic processing products was indicated to be crucial for the formation of an active catalytic replication complex. (ii) Synthesis of negative-strand intermediate and progeny positive-strand RNA was observed to be strictly coupled with all functional DI9c ORF derivatives. NS3 to NS5B were hence suggested to play a pivotal role even during early steps of the viral replication pathway. (iii) Mutations in the NS3 and NS4B units which generated nonfunctional or less functional RNAs were determined to be cis dominant. Likewise, lethal alterations in the NS4A and NS5B regions were invariably noncomplementable. (iv) In surprising contrast, replication of functional and nonfunctional NS5A mutants could be clearly enhanced and restored, respectively. In summary, our data provide initial insights into the organization of the pestivirus replication machinery.

Animals↗
PubMed2004

mRNA molecules containing murine leukemia virus packaging signals are encapsidated as dimers.

Prior work by others has shown that insertion of psi (i.e., leader) sequences from the Moloney murine leukemia virus (MLV) genome into the 3' untranslated region of a nonviral mRNA leads to the specific encapsidation of this RNA in MLV particles. We now report that these RNAs are, like genomic RNAs, encapsidated as dimers. These dimers have the same thermostability as MLV genomic RNA dimers; like them, these dimers are more stable if isolated from mature virions than from immature virions. We characterized encapsidated mRNAs containing deletions or truncations of MLV psi or with psi sequences from MLV-related acute transforming viruses. The results indicate that the dimeric linkage in genomic RNA can be completely attributed to the psi region of the genome. While this conclusion agrees with earlier electron microscopic studies on mature MLV dimers, it is the first evidence as to the site of the linkage in immature dimers for any retrovirus. Since the Psi(+) mRNA is not encapsidated as well as genomic RNA, it is only present in a minority of virions. The fact that it is nevertheless dimeric argues strongly that two of these molecules are packaged into particles together. We also found that the kissing loop is unnecessary for this coencapsidation or for the stability of mature dimers but makes a major contribution to the stability of immature dimers. Our results are consistent with the hypothesis that the packaging signal involves a dimeric structure in which the RNAs are joined by intermolecular interactions between GACG loops.

Animals↗
PubMed2005

Dcp2 Decaps m2,2,7GpppN-capped RNAs, and its activity is sequence and context dependent.

Hydrolysis of the mRNA cap plays a pivotal role in initiating and completing mRNA turnover. In nematodes, mRNA metabolism and cap-interacting proteins must deal with two populations of mRNAs, spliced leader trans-spliced mRNAs with a trimethylguanosine cap and non-trans-spliced mRNAs with a monomethylguanosine cap. We describe here the characterization of nematode Dcp1 and Dcp2 proteins. Dcp1 was inactive in vitro on both free cap and capped RNA and did not significantly enhance Dcp2 activity. Nematode Dcp2 is an RNA-decapping protein that does not bind cap and is not inhibited by cap analogs but is effectively inhibited by competing RNA irrespective of RNA sequence and cap. Nematode Dcp2 activity is influenced by both 5' end sequence and its context. The trans-spliced leader sequence on mRNAs reduces Dcp2 activity approximately 10-fold, suggesting that 5'-to-3' turnover of trans-spliced RNAs may be regulated. Nematode Dcp2 decaps both m(7)GpppG- and m(2,2,7)GpppG-capped RNAs. Surprisingly, both budding yeast and human Dcp2 are also active on m(2,2,7)GpppG-capped RNAs. Overall, the data suggest that Dcp2 activity can be influenced by both sequence and context and that Dcp2 may contribute to gene regulation in multiple RNA pathways, including monomethyl- and trimethylguanosine-capped RNAs.

Animals↗
PubMed1999

Herpes simplex virus latency-associated transcript encodes a protein which greatly enhances virus growth, can compensate for deficiencies in immediate-early gene expression, and is likely to function during reactivation from virus latency.

Herpes simplex virus types 1 and 2 (HSV1 and HSV2) enter and reactivate from latency in sensory neurons, although the events governing these processes are little understood. During latency, only the latency-associated transcripts (LATs) are produced. However, although the LAT RNAs were described approximately 10 years ago, their function remains ambiguous. Mutations affecting the LATs have minimal effects other than a small reduction in establishment of and reactivation from latency in some cases. Mutations in putative LAT-contained open reading frames (ORFs) have so far shown no effect. The LATs consist of a large species from which smaller (approximately 2 kb), nuclear, nonlinear LATs which are abundant during latency are spliced. Thus, translation of ORFs in these smaller LATs would not usually be expected to be possible, and if expressed at all, their expression might be tightly regulated. Here we show that deregulated expression of the largest HSV1 2-kb LAT-contained ORF in various cells of neuronal and nonneuronal origin greatly enhances virus growth in a manner specific to HSV1-the HSV1 LAT ORF has no effect on the growth of HSV2. Similar results of enhanced growth were found when the HSV1 LAT ORF was constitutively expressed from within the HSV1 genome. The mechanism of LAT ORF action was strongly suggested to be by substituting for deficiencies in immediate-early (IE) gene expression (particularly ICP0), because deregulated LAT ORF expression, as well as enhancing wild-type virus growth, was also found to allow efficient growth of viruses with mutations in ICP0 or VMW65. Such viruses otherwise exhibit considerable growth defects. IE gene expression deficiencies are often the block to productive infection in nonpermissive cells and are also evident during latency. These results, which we show to be protein- rather than RNA-mediated effects, strongly suggest a function of the tightly regulated expression of a LAT ORF-encoded protein in the reactivation from HSV latency.

Animals↗
PubMed1998

9-Nitrocamptothecin inhibits tumor recrosis factor-mediated activation of human immunodeficiency virus type 1 and enhances apoptosis in a latently infected T cell clone.

Transition from latency to active replication is a crucial stage for the process of human immunodeficiency virus type 1 (HIV-1) infection and life cycle. HIV-1 replication in latently infected cells can be strongly induced by the cytokine tumor necrosis factor alpha (TNF-alpha) and the proliferation-arresting chemical sodium butyrate (NaB). We have investigated the ability of the drug 9-nitrocamptothecin (9NC), a potent cellular topoisomerase I (topo I) inhibitor currently in clinical trials in cancer patients, to regulate HIV-1 replication in latently infected lymphocytic ACH-2 cells on reactivation with either TNF-alpha or NaB. Treatment of ACH-2 cells with 9NC alone resulted in increased levels of viral transcripts, while there was a slight reduction or no change in the levels of host cell transcripts. However, pretreatment of ACH-2 cells with 9NC inhibited TNF-alpha-induced extracellular HIV-1 p24 levels up to approximately 95% and nearly 80% of the cell-associated viral RNAs. The quantitative decrease in viral products was concomitant with a decrease in cellular gene expression and induction of apoptosis in the host cells. 9NC blocked the infected cells at the boundary of the S and G2 phases, resulting in an accelerated apoptosis that was further enhanced with TNF-alpha treatment. Similar results were observed following concurrent exposure to TNF-alpha and 9NC, but 9NC failed to inhibit upregulation of HIV-1 mRNA in ACH-2 cells exposed to TNF-alpha before 9NC treatment. Further, 9NC had no inhibitory effect on NaB-induced apoptosis and upregulation of HIV-1 mRNA expression regardless of whether 9NC and NaB were used concurrently or in various treatment sequences. In uninfected lymphocytic CEM cells derived from a common parental cell line, a slight downregulation of cellular gene expression was detected along with low-level apoptosis. These results demonstrate that 9NC impairs TNF-alpha-induced, but not NaB-induced, HIV-1 activation, and suggest a means of inhibiting active HIV-1 viremia arising as a result of elevated TNF-alpha levels.

Antineoplastic Agents↗
PubMed1999

Barley yellow dwarf virus RNA requires a cap-independent translation sequence because it lacks a 5' cap.

A 3' translation enhancer (3'TE) sequence that facilitates cap-independent translation is located near the 3' end of barley yellow dwarf luteovirus RNA. Here, we show that the 3'TE is required for translation of the viral genome and thus for viral replication. Antisense inhibition showed that the 3'TE has significant secondary structure and is required for translation of virion RNA from infected plants and uncapped genomic transcripts but not for translation of capped transcripts. Direct end-labeling of RNAs verified the absence of a 5' modification on virion RNA. Thus barley yellow dwarf virus differs from related viruses by having neither a genome-linked protein nor a 5' cap.

Enhancer Elements, Genetic↗
PubMed2005

Hypoxic stress enhances osteoclast differentiation via increasing IGF2 production by non-osteoclastic cells.

Development of bone depends on a continuous supply of bone-degrading osteoclasts. Although several factors such as cytokines and integrins have been shown to be important for osteoclast recruitment, their mechanism of action is poorly understood. In this study, we demonstrated the enhancement of osteoclast formation by hypoxia and investigated the molecular mechanisms involved. Primary mouse bone marrow cells were cultured in normoxic and hypoxic conditions, and RNA was prepared from each group of cells. Total RNAs were applied to a DNA microarray analysis and then RT-PCR was performed to confirm the microarray data. The most interesting finding of our microarray analysis was upregulation of insulin-like growth factor 2 (IGF2) and stromal cell-derived factor 1 (SDF1) under hypoxic conditions. RT-PCR analysis revealed that IGF2 expression was markedly upregulated in the non-osteoclastic cells. The addition of exogenous IGF2 increased the number of osteoclastic TRAP-positive multinuclear cells formed under normoxic conditions, whereas the addition of exogenous SDF1 did not change osteoclast formation. These results suggest that the upregulation of IGF2 derived from non-osteoclastic cells might be a crucial factor for osteoclast differentiation.

Adaptation, Physiological↗
PubMed2005

Identification of potential biomarkers of Peyronie's disease.

AIM: To identify proteins that are differentially expressed in cells derived from normal and diseased tunica albuginea (TA) as related to Peyronie's disease (PD). METHODS: Cells with characteristics of fibroblasts were isolated from two tissue sources. Those from the plaque of patients with PD were designated as PT cells, and those from the normally-appearing TA of the same patients were designated as NT cells. Messenger RNAs of these cells were analyzed by real-time polymerase chain reaction (RT-PCR) for the expression of monocyte chemoattractant protein 1 (MCP-1). Crude protein lysates were analyzed by surface-enhanced laser desorption/ionization mass spectrometry (SELDI-MS) with IMAC30-Cu, CM10, and H50 chips. Each lysate was then separated into six fractions, which were further analyzed by SELDI-MS. RESULTS: RT- PCR analysis showed that PT cells expressed higher levels of MCP-1 than their counterpart NT cells. SELDI-MS analysis showed that the crude protein lysates of all four cell strains produced similar and reproducible protein profiles on IMAC30-Cu and CM10 chips. Additional SELDI-MS analyses with the fractionated lysates detected three proteins of 11.6 kDa, 14.5 kDa, 22.6 kDa that were upregulated in PT cells and two proteins of 6.3 kDa and 46.9 kDa that were downregulated in PT cells. CONCLUSION: MCP-1, which is often involved in tissue fibrosis, was expressed at higher levels in PT than that in NT cells. Five potential biomarkers for PD were identified by SELDI-MS analysis.

Base Sequence↗
PubMed1984

Pausing and premature termination of human RNA polymerase II during transcription of adenovirus in vivo and in vitro.

The major late transcriptional unit of adenovirus type 2 has served as a model for studying transcription in eukaryotes. We report that pausing and premature termination are intrinsic to the transcription of this transcriptional unit by RNA polymerase II. In vivo and in isolated nuclei, transcription pauses at discrete sites proximal to the initiation site and can prematurely terminate at nucleotide 175 and possibly also at nucleotide 120. The prematurely terminated RNAs are not associated with the transcription complexes and accumulate in the cell nucleus in vivo, whereas paused RNAs remain associated with the transcription complexes and elongate into full-length transcripts. Pausing is also reproduced in the transcription complexes in a soluble system. 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole enhances pausing but not premature termination, and its action is reversible. The proposed premature termination site at nucleotide 175 in adenovirus type 2 bears sequence homology to the tR1 site in coliphage lambda.

Adenoviruses, Human↗
PubMed1998

Transcript analysis of the pcbABC genes encoding the antenna apoproteins in the photosynthetic prokaryote, Prochlorothrix hollandica.

The tightly linked pcbABC genes encode the chlorophyll a/b-binding apoproteins in the oxygenic photosynthetic prokaryote Prochlorothrix hollandica. Northern blotting experiments employing gene-specific DNA probes have identified a complex pattern of transcription from the pcb region. A large 4.4-kb transcript detected in cultures maintained in high light, low light and in darkness results from the cotranscription of all three genes, whereas pcbAB, pcbBC and individual pcbA, B, and C mRNAs are similarly detected in all light regimes. The half lives of the RNAs vary from 15 min for the pcbABC transcript, to over 60 min for the pcbA and pcbC mRNAs. The lack of identifiable promoter sequences other than the region upstream from pcbA, plus the enhanced stability of the individual single gene transcripts, suggest that the smaller RNA species arise from processing of larger transcripts. Transcription and mRNA turnover occurs largely independent of light intensity, in contrast to what is seen in most other phototrophs, in which light influences the accumulation of antenna apoprotein gene mRNAs.

Apoproteins↗
PubMed1997

Effects of reduced maternal lipoprotein-cholesterol availability on placental progesterone biosynthesis in the baboon.

Maternal low density lipoprotein (LDL) is the principal source of cholesterol substrate for progesterone biosynthesis in the primate placental syncytiotrophoblast. The relationship of LDL-cholesterol availability and other potential cholesterol-yielding pathways to placental progesterone production have not, however, been demonstrated in vivo in a nonhuman primate. Therefore, maternal peripheral lipoprotein-cholesterol and progesterone concentrations were determined in blood samples obtained by venipuncture, from day 72 until day 100, from pregnant baboons (Papio sp) that were either untreated (n = 4) or treated (n = 3) with the inhibitor of hepatic lipoprotein production, 4-aminopyrazolo [3-4-d]pyrimidine (4-APP, 10 mg/kg BW) on days 98-99 of pregnancy (term = 184 days). Although LDL-cholesterol and progesterone levels remained unchanged in untreated animals, LDL-cholesterol concentrations were 9-fold lower (P < 0.005) in baboons receiving 4-APP than in untreated baboons 2 days following initial administration. Commensurate progesterone levels were 3.5-fold lower (P < 0.03) in 4-APP-treated baboons than in untreated baboons. RT-PCR was used to approximate relative changes in transcription of messengers RNAs (mRNAs) for selected cholesterol-sensitive pathways in placental tissue collected on day 100. Thus, expression of mRNAs for LDL receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase appeared enhanced, whereas acyl-coenzyme A:cholesterol acyl transferase (ACAT) mRNA was diminished in syncytiotrophoblast-enriched cell fractions as a result of 4-APP administration. No relative differences in mRNAs were apparent in whole placental villous tissue, however, as a result of 4-APP treatment. In summary, this experiment demonstrates a significant decline in progesterone production elicited by maternal LDL-cholesterol withdrawal, and attests to the efficacy of 4-APP administration during baboon pregnancy. These results also suggest a commensurate regulation of cholesterol-sensitive pathways in primate syncytiotrophoblast. However, no relative differences were apparent in mRNA levels for LDL receptor, HMG-CoA and ACAT in whole placental villous tissue as a result of LDL-cholesterol withdrawal, which may suggest potential disparities in the mechanisms regulating cholesterol homeostasis in steroidogenically active syncytiotrophoblasts vs. those in proliferative nonendocrine placental constituents.

Adenine↗
PubMed2005

Using microarrays to facilitate positional cloning: identification of tomosyn as an inhibitor of neurosecretion.

Forward genetic screens have been used as a powerful strategy to dissect complex biological pathways in many model systems. A significant limitation of this approach has been the time-consuming and costly process of positional cloning and molecular characterization of the mutations isolated in these screens. Here, the authors describe a strategy using microarray hybridizations to facilitate positional cloning. This method relies on the fact that premature stop codons (i.e., nonsense mutations) constitute a frequent class of mutations isolated in screens and that nonsense mutant messenger RNAs are efficiently degraded by the conserved nonsense-mediated decay pathway. They validate this strategy by identifying two previously uncharacterized mutations: (1) tom-1, a mutation found in a forward genetic screen for enhanced acetylcholine secretion in Caenorhabditis elegans, and (2) an apparently spontaneous mutation in the hif-1 transcription factor gene. They further demonstrate the broad applicability of this strategy using other known mutants in C. elegans,Arabidopsis, and mouse. Characterization of tom-1 mutants suggests that TOM-1, the C. elegans ortholog of mammalian tomosyn, functions as an endogenous inhibitor of neurotransmitter secretion. These results also suggest that microarray hybridizations have the potential to significantly reduce the time and effort required for positional cloning.

Journal Article↗
PubMed2006

PUF proteins bind Pop2p to regulate messenger RNAs.

PUF proteins, a family of RNA-binding proteins, interact with the 3' untranslated regions (UTRs) of specific mRNAs to control their translation and stability. PUF protein action is commonly correlated with removal of the poly(A) tail of target mRNAs. Here, we focus on how PUF proteins enhance deadenylation and mRNA decay. We show that a yeast PUF protein physically binds Pop2p, which is a component of the Ccr4p-Pop2p-Not deadenylase complex, and that Pop2p is required for PUF repression activity. By binding Pop2p, the PUF protein simultaneously recruits the Ccr4p deadenylase and two other enzymes involved in mRNA regulation, Dcp1p and Dhh1p. We reconstitute regulated deadenylation in vitro and demonstrate that the PUF-Pop2p interaction is conserved in yeast, worms and humans. We suggest that the PUF-Pop2p interaction underlies regulated deadenylation, mRNA decay and repression by PUF proteins.

3' Untranslated Regions↗
PubMed1995

Improved accumulation and activity of ribozymes expressed from a tRNA-based RNA polymerase III promoter.

RNA polymerase III (pol III) transcripts are abundant in all cells. Therefore, pol III promoters may be ideal for expressing high levels of exogenous RNAs, such as antisense RNAs, decoy RNAs and ribozymes, in many different cell types. We have improved accumulation of recombinant RNAs expressed from a human meti tRNA-derived pol III promoter > 100-fold by modifying the 3' terminus of the transcripts to hybridize to the 5' terminus. This terminal duplex includes the 8 nt leader sequence present in the primary wild-type meti tRNA transcript that is normally removed during processing to the mature tRNA. Expression of an anti-HIV ribozyme was analyzed in cells stably transduced with retroviral vectors encoding pol III transcription units containing this modification. High accumulation of recombinant pol III ribozyme transcripts was observed in all cell lines tested. Due to the enhanced transcript accumulation, ribozyme cleavage activity was readily detectable in total RNA extracted from stably transduced human T cell lines. One pol III transcription unit, termed 'TRZ', was optimized further for ribozyme cleavage activity. The improved pol III transcription units reported here may be useful for expressing a variety of functional and therapeutic RNAs.

Base Sequence↗