Proximity to the Promoter and Terminator Regions Regulates the Transcription Enhancement Potential of an Intron

Abstract

An evolutionarily conserved feature of introns is their ability to enhance expression of genes that harbor them. Introns have been shown to regulate gene expression at the transcription and post-transcription level. The general perception is that a promoter-proximal intron is most efficient in enhancing gene expression and the effect diminishes with the increase in distance from the promoter. Here we show that the intron regains its positive influence on gene expression when in proximity to the terminator. We inserted ACT1 intron into different positions within IMD4 and INO1 genes. Transcription Run-On (TRO) analysis revealed that the transcription of both IMD4 and INO1 was maximal in constructs with a promoter-proximal intron and decreased with the increase in distance of the intron from the promoter. However, activation was partially restored when the intron was placed close to the terminator. We previously demonstrated that the promoter-proximal intron stimulates transcription by affecting promoter directionality through gene looping-mediated recruitment of termination factors in the vicinity of the promoter region. Here we show that the terminator-proximal intron also enhances promoter directionality and results in compact gene architecture with the promoter and terminator regions in close physical proximity. Furthermore, we show that both the promoter and terminator-proximal introns facilitate assembly or stabilization of the preinitiation complex (PIC) on the promoter. On the basis of these findings, we propose that proximity to both the promoter and the terminator regions affects the transcription regulatory potential of an intron, and the terminator-proximal intron enhances transcription by affecting both the assembly of preinitiation complex and promoter directionality.

Keywords: gene architecture; gene looping; gene regulation; intron; promoter directionality; splicing; transcription; yeast.

FIGURE 1

Deletion of the terminator-proximal intron of YPL109C reduces both the transcription and looped conformation of the gene. (A) Schematic depiction of YPL109C with the intron present (yellow) and intron deleted (blue) indicating the position of primers used for RT-PCR, TRO and 3C analyses. (B) Quantification of total RNA level of YPL109C measured by RT-PCR approach in the presence of the terminator-proximal intron (yellow bar) and the absence of intron (blue bar). The 18S RNA signal was used as a normalization control. (C) Quantification of nascent RNA level of YPL109C measured by TRO approach in the presence of the terminator-proximal intron (yellow bar) and the absence of intron (blue bar). The 18S RNA signal was used as a normalization control. (D) Gene looping of YPL109C measured in terms of P1T1 PCR signal by 3C approach in the presence (yellow bar) and absence (blue bar) of an intron. The F1R1 PCR represents the loading control that was used to ensure equal amounts of template DNA were present in each 3C PCR reaction. The values and error bars for each condition indicate the mean ± standard deviation. Statistical significance (p-values) was determined using the two-tailed paired Student’s t-test. Four asterisks (****) indicate p value smaller than 0.0001 (p ≤ 0.0001). (E) RT-PCR analysis of YPL109C mRNA in the construct with intron (+ intron) and without intron (− intron). Genomic DNA PCR of intron-containing strain indicates the position of unspliced transcripts. The expected positions of spliced and unspliced transcripts obtained by RT-PCR approach for each construct is indicated.

FIGURE 2

Position of an intron within the gene impacts gene looping of IMD4. (A) Schematic depiction of IMD4 gene without intron (-intron, white), and with ACT1 intron inserted near the promoter (P-intron, red), in the middle of the gene (M-intron, green) and in the proximity of the terminator (T-intron, purple) region. “p” and “T” stand for the promoter and terminator respectively. Vertical lines show position of restriction cut sites, while P1, T1, F1 and R1 are the position of primers used to amplify 3C PCR products. (B) 3C analysis of IMD4 in the absence of an intron (white bar), and with the intron inserted near the promoter (red bar), in the middle (green bar) and toward the terminator region of the gene (purple bar). P1T1 PCR represents gene looping signal, while F1R1 PCR represents the loading control that was used to ensure equal amounts of template DNA were present in each 3C PCR reaction. Results presented here represent three biological replicates and six technical replicates. The values and error bars for each condition indicate the mean ± standard deviation. Statistical significance (p-values) was determined using the two-tailed paired Student’s t-test. Two asterisks (**) signify a p value equal to or smaller than 0.01 (p ≤ 0.01). Three asterisks (***) indicate p value equal/smaller than 0.001 (p ≤ 0.001). Four asterisks (****) indicate p value equal/smaller than 0.0001 (p ≤ 0.0001). Any non-significant difference is represented by ns (p > 0.05).

FIGURE 3

Position of the intron in INO1 gene influences the transcription of the gene. (A) Schematic representation of INO1 gene without intron (1) and with the ACT1 intron inserted at five different positions along the body of the gene (2–6). “p” and “T” stand for the promoter and terminator respectively. (B) Quantification of TRO data of INO1 gene in the absence of an intron (#1) and the presence of an intron at 100 bp position (#2), 500 bp (#3), 800 bp (#4), 1,400 bp (#5), and 10 bp downstream of (TGA + 10) (#6). The transcript levels of 18S were used as the normalization control.

FIGURE 4

Both the promoter and terminator proximal introns enhance transcription and promoter directionality of IMD4 gene. (A) Schematic depiction of IMD4 gene without intron (-intron, white), and with ACT1 intron inserted near the promoter (P-intron, red), in the middle of the gene (M-intron, green) and in the proximity of the terminator (T-intron, purple) region. “p” and “T” stand for the promoter and terminator respectively. (B) TRO analysis of IMD4 in the absence of an intron (white bar), and with the intron inserted near the promoter (red bar), in the middle (green bar) and toward the terminator region of the gene (purple bar). (C) Measurement of mRNA and uaRNA levels of IMD4 in the 400 bp upstream (uaRNA) and downstream (mRNA) promoter proximal region by strand-specific TRO approach in the absence of an intron (white bar for mRNA, diagonal black pattern bar for uaRNA), and with the intron inserted near the promoter (red bar for mRNA, diagonal red pattern bar for uaRNA), in the middle (green bar for mRNA, diagonal green pattern bar for uaRNA) and toward the terminator region of the gene (purple bar for mRNA, diagonal purple pattern bar for uaRNA). (D) Directionality index of IMD4 without intron (white bar) and with the intron present at three different positions (red, green and purple bars) by comparing levels of mRNA vs. uaRNA produced. The 18S signal was used as a normalization control in these experiments. Results presented here represent three biological replicates and six technical replicates. The values and error bars for each condition indicate the mean ± standard deviation. Statistical significance (p-values) was determined using the two-tailed paired Student’s t-test. One asterisk (*) signifies a p value equal to or smaller than 0.05 (p ≤ 0.05). Any non-significant difference is represented by ns (p > 0.05).

FIGURE 5

Position of intron affects TFIIB and TFIIH promoter occupancy for IMD4 gene. (A) Schematic depiction of IMD4 without intron and with the ACT1 intron inserted near the promoter (P-intron), in the middle (M-intron) and near the terminator region (T-intron) of the gene. “p” and “T” stand for the promoter and terminator respectively. (B) Schematic depiction of IMD4 gene with intron indicating the position of primer pairs used in ChIP analysis. (C) ChIP analysis showing crosslinking of HA-tagged TFIIB to the promoter and downstream regions of IMD4 with promoter-proximal intron (red bar), middle intron (green bar), terminator-proximal intron (purple bar) and without intron (white bar). (D) ChIP analysis showing crosslinking of TAP-tagged Ccl1 subunit of TFIIH to the promoter and downstream coding regions of IMD4 with promoter-proximal intron (red bar), middle intron (green bar), terminator-proximal intron (purple bar) and without intron (white bar). The Input signal, representing DNA prior to immunoprecipitation, was used as normalization control. ChIP results presented here represent three biological replicates and six technical replicates. The values and error bars for each condition indicate the mean ± standard deviation. Statistical significance (p-values) was determined using the two-tailed paired Student’s t-test. One asterisk (*) signifies a p value equal to or smaller than 0.05 (p ≤ 0.05). Two asterisks (**) signify a p value equal to or smaller than 0.01 (p ≤ 0.01). Any non-significant difference is represented by ns (p > 0.05).

FIGURE 6

Presence of an intron does not affect termination of transcription of IMD4. (A) Schematic depiction of IMD4 with intron inserted near the promoter (P-intron), middle of the gene (M-intron) and near the terminator region (T-intron), showing the position of primers 1, 2, 3, and four used in strand-specific TRO analysis. “p” and “T” stand for the promoter and terminator respectively. (B) Strand-specific TRO analysis showing that the polymerase did not readthrough in the regions 2, 3, and four downstream of the terminator signal of IMD4 in the construct P-intron (red bar), M-intron (green bar) and T-intron (purple bar). The 18S signal was used as a normalization control in these experiments. Results presented here represent three biological replicates and six technical replicates. The values and error bars for each condition indicate the mean ± standard deviation. Statistical significance (p-values) was determined using the two-tailed paired Student’s t-test. One asterisk (*) signifies a p value equal to or smaller than 0.05 (p ≤ 0.05). Two asterisks (**) signify a p value equal to or smaller than 0.01 (p ≤ 0.01). Three asterisks (***) indicate p value equal/smaller than 0.001 (p ≤ 0.001). Four asterisks (****) indicate p value equal/smaller than 0.0001 (p ≤ 0.0001). Any non-significant difference is represented by ns (p > 0.05). (C) Readthrough Index (RTI) was calculated by dividing the TRO signal intensity beyond the 3′ end of the gene (region 4) with the signal value before the 3′ end within the coding region (region 1).