Co-transcriptional gene regulation in eukaryotes and prokaryotes

Abstract

Many steps of RNA processing occur during transcription by RNA polymerases. Co-transcriptional activities are deemed commonplace in prokaryotes, in which the lack of membrane barriers allows mixing of all gene expression steps, from transcription to translation. In the past decade, an extraordinary level of coordination between transcription and RNA processing has emerged in eukaryotes. In this Review, we discuss recent developments in our understanding of co-transcriptional gene regulation in both eukaryotes and prokaryotes, comparing methodologies and mechanisms, and highlight striking parallels in how RNA polymerases interact with the machineries that act on nascent RNA. The development of RNA sequencing and imaging techniques that detect transient transcription and RNA processing intermediates has facilitated discoveries of transcription coordination with splicing, 3'-end cleavage and dynamic RNA folding and revealed physical contacts between processing machineries and RNA polymerases. Such studies indicate that intron retention in a given nascent transcript can prevent 3'-end cleavage and cause transcriptional readthrough, which is a hallmark of eukaryotic cellular stress responses. We also discuss how coordination between nascent RNA biogenesis and transcription drives fundamental aspects of gene expression in both prokaryotes and eukaryotes.

Figure 1.. Organization of gene expression machineries and co-transcriptional processes in pro- and eukaryotic cells.

(a-b) Schematic of prokaryotic and eukaryotic cells drawn to scale (e.g., E. coli 2 μm long, HeLa cell 30 μm long), showing cellular organization with membrane-bound (nuclear envelope in eukaryotes) and membrane-less compartments (e.g., nucleolus, speckle, Cajal and P bodies, and stress granule). Gene expression is spatially organized in prokaryotes, with the nucleoid containing DNA and associated proteins in the center. E. coli cell and line plot based on imaging data^,,,,. Black arrows indicate molecular exchange by diffusion (a) and nuclear export (b). (c-d) Co-transcriptionality of gene expression processes in pro- and eukaryotes enables cross-regulation, indicated by light arrows. Multiple (possibly overlapping) coding sequences (CDS) can be encoded within one operon in prokaryotes. In eukaryotes, a single transcription unit includes exons and introns, yielding a single CDS after pre-mRNA splicing. Alternative 5′ and 3′ ends, as well as cap(-like) moieties and RNA folding are common in both systems, mechanistic differences/commonalities given as bullet points within the figure. Translation initiates co-transcriptionally in prokaryotes, but the proximity of the ribosome to RNA polymerase varies: 1^st RNA polymerase and ribosome resemble traditional view in E. coli, 2^nd RNA polymerase and ribosome reflect emerging view in different species and conditions^,. In eukaryotes, pre-mRNA splicing often occurs co-transcriptionally.

Figure 2.. Splicing estimates measured in terms of (a) Pol II position or (b) time.

Distance-based methods measure the position of Pol II where upstream exon-exon ligation is detected (distance “0” is the position of the 3′ splice site, which is required for the second step of splicing)^,,,,–,,–. Time-based methods calculate intron half-life or the time required for splicing completion^,,,–. The values along the gene ruler or timeline represent the half-maximum or a similar value if the half-maximum was not reported. Dashed lines in panel (a) separate rapid and delayed co-transcriptional splicing and post-transcriptional splicing. Rapid co-transcriptional splicing occurs while Pol II is transcribing early in the gene (<1.5 kb), delayed co-transcriptional splicing occurs while Pol II is transcribing late in the gene (>1.5 kb), and post-transcriptional splicing occurs after Pol II has completed transcription of the gene. Each study is color-coded according to the organism used to obtain the estimate (see key). This figure has been updated and also modified (with permission) from Refs^, in recognition of the fact that splicing estimates obtained in terms of distance or time cannot yet be directly compared, due to varying transcription elongation rates within gene bodies (see text).

Figure 3.. Interplay between RNA processing and Pol II transcription.

(a) Components of the RNA processing machinery interact with the Pol II C-Terminal Domain (CTD) (capping enzymes, cyan; cleavage and polyadenylation factors, green) or other areas of Pol II (U1 snRNP, orange; spliceosome, purple). CTD phosphorylation is marked with an “x”. (b) In addition to telescripting, in which U1 snRNP binding to AU-rich nascent RNA prevents usage of premature cleavage and polyadenylation (PCPA) sites, U1 snRNP binding to the Pol II large subunit helps to propel Pol II elongation through introns six times faster. Transcriptional pausing is associated with promoter-proximal regions where capping occurs as well as with the PAS where 3′ end cleavage occurs. Pause sites must be distinguished from changing elongation rates that are faster over AT-rich introns than over CpG-rich regions and GC-rich exons. (c) These variations in elongation rates and pausing at specific sites creates overall higher Pol II densities (PRO-seq) over exons compared to introns, although more nascent RNA is produced (TT-seq) over introns due to faster RNA synthesis. (d) GC content is greater in exons and lower in introns, allowing intron/exon structure to be interpreted as a function of GC-richness.

Figure 4.. Splicing outcomes involving transcriptional readthrough and/or stress.

(a) In all-or-none (pre-)mRNA processing, transcripts synthesized by RNA polymerase II (Pol II) are either all spliced with efficient 3′ end cleavage carried out by the cleavage and polyadenylation complex (CPC) (left) or all unspliced with transcriptional readthrough (right). Recruitment of positive splicing factors may contribute to “all” transcripts, while prolonged binding of splicing inhibitory factors may contribute to “none” transcripts. (b) In tomatoes, two temperature-dependent RNA structures can form at the 3′ splice site (3′SS) of the second intron in HsfA2 pre-mRNA. Under non-stress conditions (left), an elongated RNA structure forms, which orients the polypyrimidine tract (PPT) and 3′SS for U2 snRNP recognition and results in a fully spliced product. Under heat stress (right), a smaller RNA structure is favored, which exposes the PTT and potentially allows for binding of a splicing silencer or polypyrimidine tract-binding protein (PTB). This results in retention of intron 2. (c) Under non-stress conditions (top), Pol II transcribes until it reaches the transcription termination site, releasing spliced and polyadenylated mRNAs. Under stress conditions (bottom), Pol II continues transcribing into intergenic regions and downstream “read-in genes”, producing downstream-of-gene (DoG) transcripts in which the read-in genes are unspliced^,.