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Title: Enhancement of Transcription by a Splicing-Competent Intron Is Dependent on Promoter Directionality}
Article:
Abstract:
Enhancement of transcription by a splicing-competent intron is an evolutionarily conserved feature among eukaryotes. The molecular mechanism underlying the phenomenon, however, is not entirely clear. Here we show that the intron is an important regulator of promoter directionality. Employing strand-specific transcription run-on( TRO) analysis, we show that the transcription of mRNA is favored over the upstream anti-sense transcripts( uaRNA) initiating from the promoter in the presence of an intron. Mutation of either the 5′ or 3′ splice site resulted in the reversal of promoter directionality, thereby suggesting that it is not merely the 5′ splice site but the entire splicing-competent intron that regulates transcription directionality. ChIP analysis revealed the recruitment of termination factors near the promoter region in the presence of an intron. Removal of intron or the mutation of splice sites adversely affected the promoter localization of termination factors. We have earlier demonstrated that the intron-mediated enhancement of transcription is dependent on gene looping. Here we show that gene looping is crucial for the recruitment of termination factors in the promoter-proximal region of an intron-containing gene. In a looping-defective mutant, despite normal splicing, the promoter occupancy of factors required for poly( A)-dependent termination of transcription was compromised. This was accompanied by a concomitant loss of transcription directionality. On the basis of these results, we propose that the intron-dependent gene looping places the terminator-bound factors in the vicinity of the promoter region for termination of the promoter-initiated upstream antisense transcription, thereby conferring promoter directionality.
Introduction:
Although introns were discovered more than four decades ago, their precise physiological role in biological systems still remains an enigma[1, 2]. One of the evolutionarily conserved functions of introns in eukaryotes is in regulation of the mRNA output of a gene[2–6]. The promoter-proximal introns often stimulate transcription of genes that harbor them[4, 7–13]. This phenomenon of enhancement of transcription by a splicing-competent intron is called ‘intron-mediated enhancement of transcription’( IME)[1, 2, 4, 6, 14]. The discovery of IME coincided with the development of cDNA technology. It was observed that the expression of the cDNA version of a gene is much less efficient than its native intron-containing counterpart in transfected mammalian cell lines[12, 15]. It was soon realized that the effect of an intron on transcription is a general feature of all eukaryotic organisms, including yeast, flies, worms, plants and humans[4]. Despite the ubiquity, the molecular mechanism underlying the phenomenon remains elusive even more than 25 years after its initial discovery. Although less than 5% of genes in budding yeast contain introns, the intron-containing genes contribute nearly 28% of mRNA produced in yeast cells[3, 16]. We previously demonstrated that the intron-mediated enhancement of transcription in yeast involves gene looping, which is the physical interaction of the promoter and terminator regions of a gene in a transcription-dependent manner[10]. How the intron-facilitated looped gene architecture brings about enhancement of transcription, however, was not clear. A clue came when our laboratory and others demonstrated that gene looping confers directionality to the promoter-initiated transcription[17, 18]. The eukaryotic promoters and terminators are generally located in nucleosome free regions. Genomewide analysis has revealed that the promoters of most RNAPII-transcribed genes are bidirectional[19–27]. The transcription initiates in both the sense and upstream antisense directions from these promoters. Transcription in sense direction produces mRNA, while upstream antisense transcription generates non-coding transcripts called uaRNA( upstream antisense RNA) or PROMPT( Promoter upstream transcript)[19, 21, 26]. Transcription of the mRNA continues till the polymerase reaches the 3′ end of the gene, whereas synthesis of uaRNA is terminated when the transcript is just a few hundred to a thousand kilobase long[20]. This phenomenon is referred to as ‘promoter directionality’[28]. It is generally believed that the uaRNA synthesis in yeast is terminated in a poly( A)-independent manner by the Nrd1-Nab3-Sen1 complex[29, 30]. In mammalian systems, however, uaRNA transcription is terminated by the same cleavage and polyadenylation machinery that stops mRNA synthesis at the 3′ end of a gene in a poly( A)-dependent manner[19, 21]. A number of reports suggest that the components of cleavage and polyadenylation machinery are involved in the termination of yeast uaRNA transcription as well[17, 18, 29–32]. In both yeast and higher eukaryotes, uaRNA is immediately degraded by the RNA surveillance machinery. Because of their short half-life, yeast uaRNA species are often referred to as cryptic unstable transcripts( CUTs)[23, 24]. In mammalian cells, the asymmetric distribution of poly( A) site and U1 snRNA-binding sites in the promoter-proximal region is believed to contribute to transcription directionality[19, 21]. In budding yeast, however, gene looping has been shown to confer promoter directionality[17, 18]. How gene looping enhances transcription directionality, however, is not clear. Here we show that the transcription directionality of a subset of genes in yeast is dependent on the presence of a splicing-competent intron. The intron facilitates the recruitment of CF1, CPF and Rat1 termination complexes in the vicinity of the promoter region. We provide evidence that the intron-dependent gene looping facilitates the recruitment of termination factors near the promoter region. The recruited termination factors selectively terminate uaRNA synthesis, thereby conferring directionality to the promoter-initiated transcription.
Discussion:
Our published results suggest that it is either the transcription activator or the presence of an intron that facilitates transcription-dependent gene looping[10, 42]. The non-intronic genes are dependent on gene specific transcription activators for gene looping[42]. The intron-containing genes, however, require a splicing-competent intron to assume a looped gene conformation during transcription[10]. The promoter directionality of RNAPII-transcribed genes in budding yeast has been shown to be dependent on gene looping[17, 18]. Employing looping defective mutants, it was demonstrated that there is an increase in synthesis of uaRNA at the expense of mRNA in the absence of gene looping. These experiments, however, were performed with genes that exhibited activator-dependent gene looping. Here we show that the intron-dependent gene looping, which is characterized by additional interaction of the promoter and terminator regions with the intron, also confers directionality to the promoter-bound polymerase. How gene looping conferred transcription directionality was, however, not clear from any of the previous studies[17, 18]. On the basis of results presented here, we suggest a possible molecular mechanism underlying the enhancement of transcription directionality by the looped gene architecture. We propose that the proximity of the promoter and terminator regions in the gene loop allows the terminated polymerase along with the termination factors to be released from the 3′ end in the vicinity of the promoter of the gene. This leads to an increase in the local concentration of the termination factors near the 5′ end of a gene. These termination factors can now be recruited by the polymerase engaged in upstream anti-sense transcription leading to termination of uaRNA synthesis. Our hypothesis is supported by multiple experimental analyses. First, we observed enhanced crosslinking of the components of the CF1, CPF and Rat1 termination complexes near the 5′ end of several genes in the presence of an intron when the gene is in looped conformation( Fig 3B and 3C). Second, the recruitment of termination factors in the promoter-proximal region was compromised in the looping defective sua7-1 strain( Fig 7B and 7C). This mutant effects gene looping and transcription directionality without any adverse effect on splicing( S4 and S5 Figs). Third, the promoter occupancy of the termination factors exhibited a declining trend in the absence of an intron and in the presence of a mutated introns( Fig 6B). The mutation of splice sites in the intron selectively abolishes looping of the gene under investigation without any adverse effect on global gene looping[10]. It corroborates the finding with the looping defective sua7-1 mutant, which affects gene looping on a genomewide scale and therefore can potentially have an indirect effect on promoter directionality. The overall conclusion of these results is that it is looped gene architecture that facilitates the recruitment of termination factors near the 5′ end of a gene, and the termination factors then terminate the transcription of uaRNA thereby conferring promoter directionality. In mammalian cells, asymmetric distribution of poly( A) sites and U1-binding sites has been shown to influence the recruitment of termination factors in the promoter-upstream region, which in turn terminates uaRNA synthesis[19, 21]. The transcription directionality of the mammalian β-globin gene, however, was found to be compromised in a looping defective mutant of the gene[17]. This invokes the possibility of gene looping playing a similar role in the recruitment of termination factors in the promoter-proximal region of at least a subset of mammalian genes during transcription. If gene looping enhances transcription directionality by facilitating the recruitment of termination factors in the promoter-proximal region, the next logical question is why the promoter-recruited termination factors selectively terminate uaRNA synthesis, while mRNA transcription continues unabated. We have already shown that the activator-dependent gene looping facilitates recycling of polymerase from the terminator to the promoter for transcription in sense direction[18]. The intron-dependent gene looping may have a similar effect. A logical question is why the promoter recruited polymerase in a looped gene tends to preferentially transcribe in the sense direction, while it is terminated in the upstream anti-sense direction. We hypothesize that the differential effect of termination factors on the promoter-initiated divergent transcription could be due to differential chromatin structure in the vicinity of the promoter region. It has been shown that the histone modification pattern in the regions upstream and downstream of the bidirectional promoter is markedly different. In mammalian cells, the promoter downstream region is characterized by H3K79 dimethylation[22], which is the mark of elongating polymerase. In contrast, the promoter upstream region is deficient in H3K79 dimethylation[22]. Furthermore, H3K4 is trimethylated in the promoter downstream sense direction, while the upstream antisense region is marked by H3K4 monomethylation[27]. A similar differential modification of H3K27 was recently reported around the bidirectional promoter region in a murine cell line[27]. The H3K27 was found preferentially acetylated in the promoter upstream region near the antisense transcription start site in a subset of bidirectional promoters in murine macrophages. These differential chromatin marks around the bidirectional promoter region may inhibit elongation of uaRNA transcript and facilitate their termination by the termination factors. The emerging view is that a vast majority of RNAPII-transcribed genes in yeast and mammalian systems have bidirectional promoters. The regulation of promoter directionality is critical for optimal transcription of these gene. We show a novel role of introns in yeast in regulating promoter directionality through looping-mediated recruitment of termination factors at the promoter. Less than 5% of yeast genes contain introns. In contrast, a vast majority of genes in higher eukaryotes contain introns. It is, therefore, tempting to speculate that introns might have a similar mechanistic impact on transcription directionality in higher eukaryotes as well.
Eukaryotic genes differ from their prokaryotic counterparts in having intervening non-coding sequences called introns . The precise biological role of introns in eukaryotic systems remains unclear even more than forty years after their initial discovery . One function of intron that has been remarkably conserved during evolution is their ability to enhance the transcription of genes that harbor them . How does the intron regulate transcription , however , is not known . Here we show that the intron enhances gene expression by affecting direction of the promoter-initiated transcription . In the presence of an intron , polymerase tends to transcribe the downstream coding region producing mRNA , while in the absence of a splicing-competent intron polymerase starts transcribing promoter upstream region producing upstream antisense RNA ( uaRNA ) . Intron-mediated promoter directionality was dependent on gene looping , which is the interaction off the promoter and terminator region of a gene in a transcription-dependent manner . We show that the intron-dependent gene looping facilitates the recruitment of termination factors in the promoter-proximal region . The recruited termination factors stop uaRNA synthesis thereby conferring directionality to the promoter-bound polymerase .