Small Nuclear RNA Gene Promoters Research Articles

Leukotriene B4 BLT Receptor Signaling Regulates the Level and Stability of Cyclooxygenase-2 (COX-2) mRNA through Restricted Activation of Ras/Raf/ERK/p42 AUF1 Pathway

Recent studies suggest that active resolution of the inflammatory response in animal models of arthritis may involve leukotriene B(4) (LTB(4))-dependent stimulation of "intermediate" prostaglandin production, which in turn favors the synthesis of "downstream" anti-inflammatory and pro-resolving lipoxins, resolvins, and protectins. We explored a putative mechanism involving LTB(4)-dependent control of cyclooxygenase-2 (COX-2) expression, the rate-limiting step in inflammatory prostaglandin biosynthesis. Indeed, LTB(4) potently up-regulated/stabilized interleukin-1beta-induced COX-2 mRNA and protein expression under conditions of COX-2 inhibitor-dependent blockade of PGE(2) release in human synovial fibroblasts (EC(50) = 16.5 + or - 1.7 nm for mRNA; 19 + or - 2.4 nm for protein, n = 4). The latter response was pertussis toxin-sensitive, and semi-quantitative reverse transcription-PCR confirmed the quantitative predominance of the BLT2 receptor. Transfection experiments, using human COX-2 promoter plasmids and chimeric luciferase-COX-2 mRNA 3'-untranslated region (3'-UTR) reporter constructs, revealed that LTB(4) exerted its stabilizing effect at the post-transcriptional level through a 116-bp adenylate/uridylate-rich sequence in the proximal region of the COX-2 3'-UTR. Using luciferase-COX-2 mRNA 3'-UTR reporter constructs and Ras/c-Raf expression and mutant constructs, we showed that the Ras/c-Raf/MEK1/2/ERK1/2 signaling pathway mediated LTB(4)-dependent COX-2 mRNA stabilization. Knockdown experiments with specific short hairpin RNAs confirmed that LTB(4) stabilization of COX-2 mRNA was apparently mediated through the RNA-binding protein, p42 AUF1. The nuclear export of p42 AUF1 was driven by c-Raf/MEK1/2/ERK1/2 signaling and sensitive to leptomycin B treatment, suggesting a CRM1-dependent mechanism. We conclude that LTB(4) may support the resolution phase of the inflammatory response by stabilizing COX-2, ensuring a reservoir of ambient pro-resolution lipid mediators.

Zebrafish U6 small nuclear RNA gene promoters contain a SPH element in an unusual location

Promoters for vertebrate small nuclear RNA (snRNA) genes contain a relatively simple array of transcriptional control elements, divided into proximal and distal regions. Most of these genes are transcribed by RNA polymerase II (e.g., U1, U2), whereas the U6 gene is transcribed by RNA polymerase III. Previously identified vertebrate U6 snRNA gene promoters consist of a proximal sequence element (PSE) and TATA element in the proximal region, plus a distal region with octamer (OCT) and SphI postoctamer homology (SPH) elements. We have found that zebrafish U6 snRNA promoters contain the SPH element in a novel proximal position immediately upstream of the TATA element. The zebrafish SPH element is recognized by SPH-binding factor/selenocysteine tRNA gene transcription activating factor/zinc finger protein 143 (SBF/Staf/ZNF143) in vitro. Furthermore, a zebrafish U6 promoter with a defective SPH element is inefficiently transcribed when injected into embryos.

Insect small nuclear RNA gene promoters evolve rapidly yet retain conserved features involved in determining promoter activity and RNA polymerase specificity

In animals, most small nuclear RNAs (snRNAs) are synthesized by RNA polymerase II (Pol II), but U6 snRNA is synthesized by RNA polymerase III (Pol III). In Drosophila melanogaster, the promoters for the Pol II-transcribed snRNA genes consist of ∼21 bp PSEA and ∼8 bp PSEB. U6 genes utilize a PSEA but have a TATA box instead of the PSEB. The PSEAs of the two classes of genes bind the same protein complex, DmSNAPc. However, the PSEAs that recruit Pol II and Pol III differ in sequence at a few nucleotide positions that play an important role in determining RNA polymerase specificity. We have now performed a bioinformatic analysis to examine the conservation and divergence of the snRNA gene promoter elements in other species of insects. The 5′ half of the PSEA is well-conserved, but the 3′ half is divergent. Moreover, within each species positions exist where the PSEAs of the Pol III-transcribed genes differ from those of the Pol II-transcribed genes. Interestingly, the specific positions vary among species. Nevertheless, we speculate that these nucleotide differences within the 3′ half of the PSEA act similarly to induce conformational alterations in DNA-bound SNAPc that result in RNA polymerase specificity.

Targeting Ku protein for sensitizing of breast cancer cells to DNA-damage

Targeting molecular components that are critically involved in the maintenance of genome stability is a promising approach for overcoming intrinsic tumor cell resistance to DNA-damaging treatments. In mammalian cells, the Ku-dependent non-homologous end-joining repair pathway is the predominant process for the repair of double-strand breaks (DSBs) in DNA. Previously, RNA aptamers were selected to efficiently block DNA-binding activity of the Ku protein in vitro. In the present study, we have tested the efficacy of RNA aptamers against the Ku protein as molecular sensitizer of MCF-7 breast carcinoma cells to DNA-damage. Toward this end, we established MCF-7 cell sublines stably expressing SC4 aptamer RNAs under the control of the human 7SL small nuclear RNA gene promoter. Vector-transfected (MCF/7SL) cells and cells stably expressing SC4 aptamers (MCF/SC4) were exposed to the anticancer drug etoposide and cellular responses to DNA-damage were evaluated. We found that the presence of RNA aptamers against Ku protein enhanced etoposide-induced growth inhibition of MCF-7 breast cancer. The SC4 aptamer-mediated sensitization of MCF-7 cells to the anticancer drug is attributable to an increased susceptibility of these cells to apoptosis. The observed effects cannot be accounted for by the differential expression levels of Ku protein in control and SC4 aptamer-expressing cells, but are rather due to augmented DNA binding-capacity of Ku protein, as demonstrated in in vitro studies. Thus, RNA aptamers against Ku protein show potential to sensitize MCF-7 breast carcinoma cells to DNA-damaging agents.

RNA polymerase II-dependent transcription in trypanosomes is associated with a SNAP complex-like transcription factor.

Spliced leader RNA transcription is essential for cell viability in trypanosomes. The SL RNA genes are expressed from the only defined RNA polymerase II-dependent promoter identified to date in the trypanosome genome. The SL RNA gene promoter has been shown by in vitro and in vivo analyses to have a tripartite architecture. The upstream most cis-acting element, called PBP-1E, is located between 70 and 60 bp upstream from the transcription start site. This essential element functions along with two downstream elements to direct efficient and proper initiation of transcription. Electrophoretic mobility-shift studies detected a 122-kDa protein, called PBP-1, which interacts with PBP-1E. This protein is the first sequence-specific, double-stranded DNA-binding protein isolated in trypanosomes. Three polypeptides copurify with PBP-1 activity, suggesting that PBP-1 is composed of 57-, 46-, and 36-kDa subunits. We have cloned the genes that encode the 57- and 46-kDa subunits. The 46-kDa protein is a previously uncharacterized protein and may be unique to trypanosomes. Its predicted tertiary structure suggests it binds DNA as part of a complex. The 57-kDa subunit is orthologous to the human small nuclear RNA-activating protein (SNAP)50, which is an essential subunit of the SNAP complex (SNAPc). In human cells, SNAPc binds to the proximal sequence element in both RNA polymerase II- and III-dependent small nuclear RNA gene promoters. These findings identify a surprising link in the transcriptional machinery across a large evolutionary distance in the regulation of small nuclear RNA genes in eukaryotes.

The Small RNA Gene Activator Protein, SphI Postoctamer Homology-binding Factor/Selenocysteine tRNA Gene Transcription Activating Factor, Stimulates Transcription of the Human Interferon Regulatory Factor-3 Gene

Many small nuclear RNA gene promoters are activated by SphI postoctamer homology (SPH)-binding factor/selenocysteine tRNA gene transcription activating factor (SBF/Staf). Whereas this transcription factor was initially identified by its ability to bind to SPH elements in such promoters, it was more recently shown to have the capacity to activate transcription of a synthetic mRNA gene promoter through a distinct activation domain. Here, we show that the human interferon regulatory factor-3 (IRF-3) gene promoter contains a functional SPH element that is bound by SBF/Staf in vitro and in transfected cells.

BRFU, a TFIIB-like factor, is directly recruited to the TATA-box of polymerase III small nuclear RNA gene promoters through its interaction with TATA-binding protein.

The human snRNA genes transcribed by RNA polymerase II (pol II) and III (pol III) have different core promoter elements. Both gene types contain similar proximal sequence elements (PSEs) but differ in the absence (pol II) or presence (pol III) of a TATA-box, which, together with the PSE, determines the assembly of a pol III-specific pre-initiation complex. BRFU is a factor exclusively required for transcription of the pol III-type snRNA genes. We report that recruitment of BRFU to the TATA-box of these promoters is TATA-binding protein (TBP)-dependent. BRFU in turn stabilizes TBP on TATA-containing template and extends the TBP footprint both upstream and downstream of the TATA element. The core domain of TBP is sufficient for BRFU.TBP.DNA complex formation and for interaction with BRFU off the template. We have mapped amino acid residues within TBP and domains of BRFU that mediate this interaction. BRFU has no specificity for sequences flanking the TATA-box and also forms a stable complex on the TATA-box of the pol II-specific adenovirus major late promoter (AdMLP). Furthermore, pol III-type transcription can initiate from an snRNA gene promoter containing an AdMLP TATA-box and flanking sequences. Therefore, the polymerase recruitment is not simply determined by the sequence of the TATA-box and immediate flanking sequences.

The Drosophila U1 and U6 Gene Proximal Sequence Elements Act as Important Determinants of the RNA Polymerase Specificity of Small Nuclear RNA Gene Promoters in Vitroand in Vivo

Transcription of genes coding for metazoan spliceosomal snRNAs by RNA polymerase II (U1, U2, U4, U5) or RNA polymerase III (U6) is dependent upon a unique, positionally conserved regulatory element referred to as the proximal sequence element (PSE). Previous studies in the organism Drosophila melanogaster indicated that as few as three nucleotide differences in the sequences of the U1 and U6 PSEs can play a decisive role in recruiting the different RNA polymerases to transcribe the U1 and U6 snRNA genes in vitro. Those studies utilized constructs that contained only the minimal promoter elements of the U1 and U6 genes in an artificial context. To overcome the limitations of those earlier studies, we have now performed experiments that demonstrate that the Drosophila U1 and U6 PSEs have functionally distinct properties even in the environment of the natural U1 and U6 gene 5'-flanking DNAs. Moreover, assays in cells and in transgenic flies indicate that expression of genes from promoters that contain the "incorrect" PSE is suppressed in vivo. The Drosophila U6 PSE is incapable of recruiting RNA polymerase II to initiate transcription from the U1 promoter region, and the U1 PSE is unable to recruit RNA polymerase III to transcribe the U6 gene.

Potent inhibition of human immunodeficiency virus type 1 (HIV-1) gene expression and virus production by an HIV-2 tat activation-response RNA decoy.

Tat activation-response region (TAR) decoys have been developed for use in gene therapy for people infected with human immunodeficiency virus type 1 (HIV-1). When a TAR RNA decoy is overexpressed, it will bind Tat, thus leaving less of this crucial protein to bind to and activate the natural transcriptional promoter of HIV-1. Previous TAR decoy constructs have used HIV-1 TAR. However, recent epidemiological and biological data began to suggest that the TAR region from the human immunodeficiency virus type 2 (HIV-2) may suppress HIV-1 transcription and hence replication. We created a vector which overexpresses TAR-2 under the control of the human U6 small nuclear RNA gene promoter and here show that the U6-TAR-2 decoy construct potently inhibits both HIV-2 and HIV-1 gene expression. Further, this decoy construct is able to markedly suppress HIV-1 replication. Thus, we have directly proven that TAR-2 can suppress HIV-1 replication and suggest that the HIV-2 TAR decoy may prove useful for combating HIV-1 infection.

Molecular cloning of a cDNA encoding human SPH-binding factor, a conserved protein that binds to the enhancer-like region of the U6 small nuclear RNA gene promoter.

Many vertebrate small nuclear RNA gene promoters contain an SPH motif in their distal control regions that can confer transcriptional stimulation by RNA polymerase II or RNA polymerase III. Using the human U6 gene SPH motif as a probe, we isolated a cDNA encoding human SPH-binding factor (hSBF) from a HeLa cell expression library. The coding region of hSBF is almost identical to ZNF143, a 626 amino acid, seven zinc finger protein of previously unknown function. Furthermore, the predicted amino acid sequence of hSBF is highly homologous to Xenopus laevis and mouse Staf proteins, that bind to SPH motifs and stimulate transcription of selenocysteine tRNA gene promoters. Recombinant hSBF expressed in vitro or from Escherichia coli bound specifically to the human U6 gene SPH motif as shown by DNase I footprinting and electrophoretic mobility shift assays using various mutant SPH sites as competitors. Antibodies prepared against recombinant hSBF inhibited assembly of native SBF-DNA complexes. Immunodepleted HeLa S100 transcription extract no longer supported elevated levels of transcription by RNA polymerase III from a U6 promoter containing an SPH motif, whereas addition of recombinant hSBF protein to the immunodepleted extract reconstituted stimulated transcription.

Pre-messenger RNA Splicing of Transcripts Synthesized from Human Small Nuclear RNA Gene Promoters

In order to explore the coupling of transcription with splicing in mammalian cells we have prepared hybrid genes in which either the human U2 promoter, recognized by RNA polymerase II, or the human U6 promoter, recognized by RNA polymerase III, was fused to an intron-containing gene segment. Neither human small nuclear RNA gene contains an intron although U6 genes from some species of yeast contain a short intervening sequence. Following transfection of human cells and analysis of specific RNAs by primer extension we found that the chimeric U2 promoter-derived transcript was efficiently spliced but the RNA polymerase III transcript driven by the U6 promoter remained unspliced. Hence, the splicing apparatus differentiates between transcripts produced from two closely related promoters that are distinguished by RNA polymerase selectivity.

The mouse U7 small nuclear RNA gene promoter functions efficiently in Xenopus oocytes

The U7 small nuclear RNA forms part of the snRNP particle which is involved in 3'-processing of replication variant histone gene transcripts. It is one of a number of minor snRNP particles whose concentration in cells are 1130th or less than those of the major snRNPs. The mechanism(s) by which the levels of the minor snRNAs and their corresponding snRNPs are regulated is presently unknown. Likely possibilities include different rates of transcription, different processing rates, different assembly rates or different stabilities. The U7 snRNA is present at 1/30th to 160th the level of the major snRNAs in sea urchin and mammals[l] and although the sea urchin U7 genes have been cloned for some time and have typical snRNA transcription elements, no-one has yet directly compared the efficiencies of major and minor snRNA gene promoters. We have recently cloned the mouse U7 small nuclear RNA gene[2] and a U7 pseudogene[3] and have been using the Xenopus oocyte injection system to study