Synthesis Of Full-length Transcripts Research Articles

Attenuation of Eukaryotic Protein-Coding Gene Expression via Premature Transcription Termination.

A complex network of RNA transcripts is generated from eukaryotic genomes, many of which are processed in unexpected ways. Here, we highlight how premature transcription termination events at protein-coding gene loci can simultaneously lead to the generation of short RNAs and attenuate production of full-length mRNA transcripts. We recently showed that the Integrator (Int) complex can be selectively recruited to protein-coding gene loci, including Drosophila metallothionein A (MtnA), where the IntS11 RNA endonuclease cleaves nascent transcripts near their 5' ends. Such premature termination events catalyzed by Integrator can repress the expression of some full-length mRNAs by more than 100-fold. Transcription at small nuclear RNA (snRNA) loci is likewise terminated by Integrator cleavage, but protein-coding and snRNA gene loci have notably distinct dependencies on Integrator subunits. Additional mechanisms that attenuate eukaryotic gene outputs via premature termination have been discovered, including by the cleavage and polyadenylation machinery in a manner controlled by U1 snRNP. These mechanisms appear to function broadly across the transcriptome. This suggests that synthesis of full-length transcripts is not always the default option and that premature termination events can lead to a variety of transcripts, some of which may have important and unexpected biological functions.

Translational control mechanism of HIV-1 tat1 mRNA

Background The Human Immunodeficiency Virus type 1 Tat protein is a major viral transactivator which stimulates the synthesis of full length transcripts by interacting with the 5’end of all nascent viral RNAs. Translation of tat mRNAscan be cap dependent. Nevertheless, in phaseG2/M ribosomes translate tat mRNAs by an alternative initiation processdepending on anlnternal Ribosome Entry Site (IRES). This initiation is auto-stimulated by Tat [1]. Recently, SRp40 and SRp55 proteins were found to promote Gag IRES-dependent translation initiation [2]. Therefore, SR and hnRNP proteins likely play a key role in regulation of both splicing and translation of HIV-1 RNA. As we previously observed a binding of hnRNP H (3), DAZAP1 and various SR proteins in the vicinity of the tat initiation codon(4), we asked whether these proteins can regulate tat mRNA translation.

FBI-1, a factor that binds to the HIV-1 inducer of short transcripts (IST), is a POZ domain protein.

The HIV-1 promoter directs the synthesis of two classes of transcripts, short, non-polyadenylated transcripts and full-length, polyadenylated transcripts. The synthesis of short transcripts is activated by a bipartite DNA element, the inducer of short transcripts or IST, located downstream of the HIV-1 transcriptional start site, while the synthesis of full-length transcripts is activated by the viral activator Tat. Tat binds to the RNA element TAR, which is encoded largely between the two IST half-elements. Upon activation by Tat, the synthesis of short RNAs is repressed. We have previously purified a factor called FBI-1 (for factor that binds to IST) whose binding to wild-type and mutated ISTs correlated well with the abilities of these ISTs to direct the synthesis of short transcripts. Here, we report the cloning of cDNAs encoding FBI-1. FBI-1 contains a POZ domain at its N-terminus and four Krüppel-type zinc fingers at its C-terminus. The C-terminus is sufficient for specific binding, and FBI-1 can form homomers through its POZ domain and, in vivo, through its zinc finger domain as well. In addition, FBI-1 associates with Tat, suggesting that repression of the short transcripts by Tat may be mediated through interactions between the two factors.

Mutations in the carboxy-terminal domain of TBP affect the synthesis of human immunodeficiency virus type 1 full-length and short transcripts similarly.

The human immunodeficiency virus type 1 promoter generates two types of RNA molecules, full-length transcripts and short transcripts. Synthesis of the short transcripts depends on the inducer of short transcripts (IST), an element located downstream of the start site. In the presence of the viral activator Tat, the synthesis of full-length transcripts is up-regulated while that of short transcripts is down-regulated. Full-length and short transcripts are probably generated by different types of transcription complexes. The first is IST independent, capable of efficient elongation, and up-regulated by Tat. The second is IST dependent, incapable of efficient elongation, and down-regulated by Tat. We have used an in vivo assay to assess the role of TBP in human immunodeficiency virus type I transcription and to test the effect of mutations in TBP on synthesis of full-length and short transcripts. We find that TBP bound to the TATA box is required for the synthesis of short and full-length transcripts as well as for Tat activation and that both yeast TBP and the carboxy-terminal domain of human TBP can replace full-length human TBP for these processes. Mutations in TBP affect the synthesis of short and full-length transcripts as well as Tat activation similarly, and these effects correlate with the previously described effects of these mutations on binding of TBP to the TBP-associated factor TAFII250 in vitro. Together, these results suggest that if short and full-length transcripts are generated by variant transcription complexes, these complexes use TBP similarly, probably as part of the TFIID complex.

Actinomycin D Stimulates the Transcription of rRNA Minigenes Transfected into Mouse Cells. Implications for the in vivo Hypersensitivity of rRNA Gene Transcription

The in vivo hypersensitivity of eukaryotic rRNA gene transcription to actinomycin D has long beenknown, but this effect could not be reproduced in model systems and its molecular mechanisms remain uncertain. We studied the action of actinomycin D using mouse rRNA minigenes (with RNA polymerase I promoter and terminator signals), carrying truncated mouse or human rDNA inserts, which are faithfully transcribed upon transient transfection into mouse cells. Low concentrations (0.01–0.08 μg/ml) of actinomycin D caused within 1–2 h a 2–7-fold stimulation of the transcription of rRNA minigenes which is inversely related to the size of the rDNA transcript. With transcripts longer than 3 kb the effect was reversed and at 4 kb a practically complete inhibition of the formation of full-length transcripts was observed, accompanied, however, by an enhanced accumulation of unfinished rDNA transcripts. The dependence of actinomycin D action on transcript length was also observed with lacZ gene segments of different size inserted into the mouse rRNA minigenes. The transcription initiation of endogenous rRNA genes was also stimulated by the low doses of actinomycin D as indicated by the enhanced synthesis of unfinished rDNA transcripts (spanning mainly the 5′ external transcribed spacer), whereas the synthesis of full-length transcripts was abolished. Removal of actinomycin D from the medium caused within 8–24 h a dramatic increase of the transcription from all rRNA minigenes tested. This stimulation was also inversely related to the size of the transcripts and varied from twofold to fivefold for the 3–4-kb transcripts to about 50–80-fold for the basic minigene transcript (395 nucleotides). The amount of endogenous aborted rDNA transcripts was also markedly increased, but the synthesis of full-length transcripts was not restored even 24 h after removal of the drug. The present results reproduce in a model cellular system the in vivo hypersensitivity of rRNA gene transcription to actinomycin D and reveal that the major factor involved is the size of the rRNA gene transcript. This effect requires only the basic rRNA gene promoter and terminator signals and does not depend on the G+C content of the RNA polymerase I transcripts. We suggest that at low concentrations, the intercalation of actinomycin D changes the conformation of DNA in the promoter region in a manner that stimulates the transcription of both endogenous and transfected rRNA genes. However, transcriptiondriven positive supercoiling of rDNA is enhanced by actinomycin D and above a critical transcript length it causes a blockage of transcription elongation and the accumulation of aborted rDNA transcripts.

Actinomycin D stimulates the transcription of rRNA minigenes transfected into mouse cells. Implications for the in vivo hypersensitivity of rRNA gene transcription.

The in vivo hypersensitivity of eukaryotic rRNA gene transcription to actinomycin D has long been known, but this effect could not be reproduced in model systems and its molecular mechanisms remain uncertain. We studied the action of actinomycin D using mouse rRNA minigenes (with RNA polymerase I promoter and terminator signals), carrying truncated mouse or human rDNA inserts, which are faithfully transcribed upon transient transfection into mouse cells. Low concentrations (0.01-0.08 micrograms/ml) of actinomycin D caused within 1-2 h a 2-7-fold stimulation of the transcription of rRNA minigenes which is inversely related to the size of the rDNA transcript. With transcripts longer than 3 kb the effect was reversed and at 4 kb a practically complete inhibition of the formation of full-length transcripts was observed, accompanied, however, by an enhanced accumulation of unfinished rDNA transcripts. The dependence of actinomycin D action on transcript length was also observed with lacZ gene segments of different size inserted into the mouse rRNA minigenes. The transcription initiation of endogenous rRNA genes was also stimulated by the low doses of actinomycin D as indicated by the enhanced synthesis of unfinished rDNA transcripts (spanning mainly the 5' external transcribed spacer), whereas the synthesis of full-length transcripts was abolished. Removal of actinomycin D from the medium caused within 8-24 h a dramatic increase of the transcription from all rRNA minigenes tested. This stimulation was also inversely related to the size of the transcripts and varied from twofold to fivefold for the 3-4-kb transcripts to about 50-80-fold for the basic minigene transcript (395 nucleotides). The amount of endogenous aborted rDNA transcripts was also markedly increased, but the synthesis of full-length transcripts was not restored even 24 h after removal of the drug. The present results reproduce in a model cellular system the in vivo hypersensitivity of rRNA gene transcription to actinomycin D and reveal that the major factor involved is the size of the rRNA gene transcript. This effect requires only the basic rRNA gene promoter and terminator signals and does not depend on the G + C content of the RNA polymerase I transcripts. We suggest that at low concentrations, the intercalation of actinomycin D changes the conformation of DNA in the promoter region in a manner that stimulates the transcription of both endogenous and transfected rRNA genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of Drosophila melanogaster F elements in vivo.

Drosophila melanogaster F elements are mobile, oligo(A)-terminated DNA sequences that probably propagate by the retrotranscription of RNA intermediates. Polyadenylated transcripts corresponding in size to full-length (4.7 kb) family members were detected in the Drosophila melanogaster Canton-S strain from 2nd larval instar to the adult stage. RNA accumulation reached a maximum in pupae. In the adult, F elements are transcribed in both sexes. F expression is directed in vivo by the intragenic promoter (Fin) located at the 5' end of F. Whole-mount hybridizations were carried out to define the site of synthesis of full-length transcripts found in the ovary. Selective RNA accumulation was not detected in the cytoplasm of any specific cell type. Stained nuclear dots were observed in nurse cells from stage 2-3 to the end of oogenesis. RNase treatment of egg chambers prior to the addition of the probe led to disappearance of the nuclear dots and appearance of a cytoplasmic hybridization signal suggesting leakage of nuclear transcripts. Transgenic lines harbouring the chloramphenicol acetyltransferase (CAT) gene under the control of the Fin promoter were obtained. In independent lines, CAT enzyme levels mirror the ontogenetic profile of F expression drawn from Northern RNA blotting data. An antisense promoter (Fout) that is located downstream from the Fin promoter and transcribe too bords the 5' end of F seems to be constitutively expressed in the fly.