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.
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Acknowledgements
The authors would like to thank J. Steitz for her many insightful comments on the manuscript and N. Said for the helpful discussions. The authors are grateful for the support from the National Institutes of Health (R01 GM112766 and R01 GM140735 to K.M.N). The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. M.S. is a recipient of a Gruber Science Fellowship, J.G. is a recipient of an NIH F31 predoctoral fellowship (F31NS129248), and L.S. is a recipient of a predoctoral fellowship from the American Heart Association (908949).
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Glossary
- 3′ splice sites
-
The 3′-most sequence of each intron, which, along with the polypyrimidine tract is recognized by U2AF.
- 5′ splice site
-
The 5′-most sequence of each intron, harbouring approximately 6 nt complementarity to the 5′ end of U1 snRNA, thereby enabling its binding for intron identification and, later during splicing, interactions with U6 snRNA.
- Alternative cleavage and polyadenylation
-
(APA). Use of alternative polyadenylation sites to generate transcript isoforms from the same gene, which can give rise to protein isoforms with distinct C-termini or contribute to transcript regulation through changes to 3′ untranslated regions.
- Alternative splicing
-
(AS). The process of generating multiple mRNA isoforms from a single gene by using different combinations of splice sites, for example, intron retention, exon skipping, and alternative 5′ splice site or 3′ splice site usage.
- Backtracking
-
Transcription pausing through diffusive movement of RNA polymerase along template DNA, maintained by feeding nascent RNA through a channel at the front of the enzyme.
- Branchpoint
-
Short cis-regulatory element containing an adenine base that is involved in lariat formation during the first step of pre-mRNA splicing; resides within the 3′ portion of the intron, usually near and upstream of another cis-regulatory element, the polypyrimidine tract.
- cis-Elements required for translation initiation
-
For most coding sequences in bacteria, these are the Shine–Dalgarno sequence, an A-rich sequence upstream of the start codon, and the start codon itself.
- Cleavage and polyadenylation
-
(CPA). Endonucleolytic cleavage followed by polyadenylation of the transcript by the CPC while Pol II continues transcription until termination downstream.
- Cleavage and polyadenylation complex
-
(CPC). Macromolecular protein complex that binds to cis-regulatory elements involved in 3′-end formation of eukaryotic mRNAs and facilitates its coupling to Pol II.
- Degradosome
-
Protein complex containing RNA degradation enzymes such as endonucleases and exonucleases, helicases and often also metabolic enzymes.
- Exon definition
-
Definition of the 3′ splice site of the intron upstream of a short exon and of the 5′ splice sites of the intron downstream of the same short exon, usually through the binding of exon sequences by splicing factors.
- Exon junction complex
-
(EJC). Macromolecular protein complex that forms at exon–exon junctions after splicing is completed and regulates post-transcriptional processes.
- Intrinsic transcription terminators
-
RNA stem–loops associated with A-tracts and U-tracts in nascent RNA that cause transcription termination of bacterial RNA polymerase.
- Nascent elongating transcript sequencing
-
(NET-seq). A method that sequences the 3′ end of all RNAs that co-purify with RNA polymerase, thereby indicating the position of all RNA polymerase holoenzymes that have initiated transcription.
- Nucleoid
-
Part of the bacterial cell plasm that contains most of the genomic DNA, nucleoid-associated structural proteins and components of the gene expression and replication machinery.
- Polyadenylation site
-
The signal for polyadenylation (AAUAAA in humans) is read by the cleavage and polyadenylation specificity factor (CPSF), upon which the cleavage and CPC is assembled and which releases the nascent transcript from the polymerase and polyadenylates it.
- Polypyrimidine tract
-
An approximately 15–20-nt-long region of pre-mRNA located near the 3′ end of introns that is rich in pyrimidine bases (that is, cytosine and uracil) and promotes spliceosome assembly by serving as a binding site for the spliceosome component U2AF.
- Polypyrimidine tract-binding protein
-
(PTB). A protein that binds to polypyrimidine tracts within introns and negatively regulates pre-mRNA splicing.
- Precision run-on sequencing
-
(PRO-eq). A method that determines the density of elongating RNA polymerases through the addition of a single biotinylated nucleotide (or BrUTP), which can be used to select the nascent RNA, obtain short reads and map the 3′-end and polymerase position.
- Recursive splicing
-
A process by which a long intron is removed in multiple smaller pieces rather than as a single unit.
- Rho
-
Bacterial homohexameric RNA helicase and chaperone whose binding to nascent RNAs, often between not-closely trailing ribosomes and the RNA polymerase, can lead to transcription termination.
- Runaway transcription
-
Transcript elongation is substantially faster than translation and, thus, the RNA polymerase outpaces ribosomes, generating ‘slack’ of nascent RNA in between the two molecular machines. First experimentally detected in B. subtilis, it also tends to occur in other bacteria.
- Serine–arginine-rich proteins
-
A family of protein splicing regulators that contain one or two RNA-recognition motifs and a serine–arginine-rich domain.
- Small RNA
-
(sRNA). Regulatory short RNAs (<200 nt) in bacteria that can affect all steps of prokaryotic gene expression, including secondary-structure formation.
- Spliceosomes
-
Multi-megadalton molecular machines composed of the small nuclear ribonucleoproteins U1, U2, U4, U5 and U6 and of associated proteins, which catalyse the removal of introns from pre-mRNA.
- Tight transcription–translation coupling
-
The first translating ribosome on nascent RNA is closely trailing the RNA polymerase, possibly even physically interacting with it; tight coupling has been the core model for co-transcriptional translation and is based on work in E. coli.
- Transcription polarity
-
Decline (5′ to 3′) of RNA signal across operons caused by transcription termination within operons at varying sites, for example, through Rho-dependent termination.
- Transient transcriptome sequencing
-
(TT-seq). A method that detects newly synthesized RNA by metabolic incorporation of 4sU in live cells and focusing on the proportion of transcripts that have been synthesized in the labelling period.
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Shine, M., Gordon, J., Schärfen, L. et al. Co-transcriptional gene regulation in eukaryotes and prokaryotes. Nat Rev Mol Cell Biol (2024). https://doi.org/10.1038/s41580-024-00706-2
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DOI: https://doi.org/10.1038/s41580-024-00706-2
