Transcriptional interference at tandem lncRNA and protein-coding genes: an emerging theme in regulation of cellular nutrient homeostasis

Abstract

Tandem transcription interference occurs when the act of transcription from an upstream promoter suppresses utilization of a co-oriented downstream promoter. Because eukaryal genomes are liberally interspersed with transcription units specifying long non-coding (lnc) RNAs, there are many opportunities for lncRNA synthesis to negatively affect a neighboring protein-coding gene. Here, I review two eukaryal systems in which lncRNA interference with mRNA expression underlies a regulated biological response to nutrient availability. Budding yeast SER3 is repressed under serine-replete conditions by transcription of an upstream SRG1 lncRNA that traverses the SER3 promoter and elicits occlusive nucleosome rearrangements. SER3 is de-repressed by serine withdrawal, which leads to shut-off of SRG1 synthesis. The fission yeast phosphate homeostasis (PHO) regulon comprises three phosphate acquisition genes - pho1, pho84, and tgp1 - that are repressed under phosphate-replete conditions by 5' flanking lncRNAs prt, prt2, and nc-tgp1, respectively. lncRNA transcription across the PHO mRNA promoters displaces activating transcription factor Pho7. PHO mRNAs are transcribed during phosphate starvation when lncRNA synthesis abates. The PHO regulon is de-repressed in phosphate-replete cells by genetic manipulations that favor 'precocious' lncRNA 3'-processing/termination upstream of the mRNA promoters. PHO lncRNA termination is governed by the Pol2 CTD code and is subject to metabolite control by inositol pyrophosphates.

Figure 1.

General scheme of transcriptional interference at tandem lncRNA and protein coding genes. See text for discussion.

Figure 2.

Regulated transcriptional interference at the budding yeast SRG1-SER3 locus in response to serine availability. (Top panel) In serine replete cells, activation of SRG1 transcription by Cha4 results in synthesis of three species of lncRNA (red wavy lines) that are processed at different poly(A) sites. Pol2 synthesizing these lncRNA will traverse the SER3 mRNA promoter. (Bottom panel) SRG1 transcription is turned off quickly in response to serine starvation, which makes the SER3 promoter available for binding to an as yet unknown activating transcription factor and hence synthesis of SER3 mRNA (blue wavy line). It is suggested that Cha4 remains associated with the SRG1 promoter during serine starvation but is unable to activate lncRNA synthesis. The putative Cha4 duplex DNA binding site is shown with the inverted repeat demarcated by arrows.

Figure 3.

Regulated transcription interference by upstream lncRNAs underlies phosphate homeostasis in fission yeast. (A) Tandem arrangements of the PHO lncRNA and mRNA genes are shown. Distance between the lncRNA and mRNA transcription start sites are indicated. The prt2, prt, and nc-tgp1 lncRNAs are depicted as red wavy lines. The pho84, pho1 and tgp1 mRNAs are blue wavy lines. (B) The nucleotide sequences and transcription start sites of the PHO lncRNA gene promoters (top strand) are shown. The TATA box and HomolD box elements are highlighted.

Figure 4.

Transcription factor Pho7 recognizes a UAS element in the PHO mRNA promoters. (A) The 738-aa Pho7 polypeptide is depicted as a horizontal bar. The internal DNA-binding domain containing the Cys₆•Zn₂ module is colored cyan. The primary structure of the minimal DBD is shown below the cartoon, with the Zn-binding cysteines indicated by dots. (B) The nucleobase sequences at the experimentally determined Pho7 binding sites in the indicated PHO mRNA promoters are aligned. Conserved positions are indicated by black dots. The Pho7 binding site consensus sequence identified by in silico analysis of genome-wide Pho7 ChIP-seq data is shown on the right. (C) Structure of Pho7 DBD bound to pho1 site 2 DNA. The Pho7–DNA complex is shown with the DNA depicted as a stick model, with an overlying transparent surface model to highlight the major and minor grooves. The Pho7 protein is rendered as a cartoon trace with cyan α-helices. The two zinc atoms are shown as green spheres and the six zinc-binding cysteines are shown as stick models and labeled. Anomalous difference density for the zinc atoms, contoured at 5σ, is shown in red mesh. The primary structure of the pho1 site 2 DNA ligand is shown at bottom. Red dots indicate DNA nucleobases that are contacted directly by Pho7 amino acids.

Figure 5.

Modulation of PHO lncRNA termination can impact PHO mRNA repression. (A) Precocious termination of lncRNA synthesis upstream of the mRNA promoter can result in de-repression of mRNA transcription. (B) Decreased termination of lncRNA synthesis can increase interference with the mRNA promoter and hyper-repress mRNA expression. See text for discussion.

Figure 6.

Enzymes of inositol pyrophosphate IP7 and IP8 metabolism in fission yeast. The chemical structures of 5-IP7 (at left, with the positions of the myo-inositol ring numbered), 1,5-IP8 (middle), and 1-IP7 (at right) are shown. Fission yeast Asp1 is a bifunctional enzyme composed of an N-terminal IPP kinase domain that converts 5-IP7 to 1,5-IP8 and a C-terminal IPP pyrophosphatase domain that converts 1,5-IP8 back to 5-IP7. Fission yeast Aps1 is an IPP pyrophosphatase that converts 1,5-IP8 to 1-IP7.