Cytoplasmic shortening of mRNA poly(A) tails represses eukaryotic gene expression by inhibiting efficient translation and committing an mRNA to decay. The CCR4–NOT deadenylase machinery interacts with sequence-specific RNA-binding proteins (RBPs), termed RNA adaptors, to target specific transcripts for deadenylation. In contrast, the PAN2–PAN3 deadenylation complex is thought to be predominantly recruited to mRNAs via interaction with the poly(A) binding protein, raising the question of whether it acts in a transcript-specific manner. Here, using biochemical reconstitution, we show that PAN2–PAN3 can also be recruited to specific RNAs via RNA adaptors, including MEX3, YTHDF and ZFP36 proteins. In cells, we find that a diverse range of RNA adaptors interact with both major deadenylation complexes. Thus, our data suggest that, in addition to CCR4-NOT, PAN2-PAN3 also contributes to the specificity of mRNA degradation and the robustness of post-transcriptional regulation of gene expression. ### Competing Interest Statement The authors have declared no competing interest. Herchel Smith PhD Studentship Medical Research Council, https://ror.org/03x94j517, MC_U105192715 European Research Council, https://ror.org/0472cxd90, 725685 National Sciences and Engineering Research Council of Canada, RGPIN-2019-06297 Canada Foundation for Innovation, https://ror.org/000az4664 Ontario Research Foundation, https://ror.org/051639p85

Poly(A) tails of newly synthesized mRNAs have uniform lengths, arising through cooperation between the cleavage and polyadenylation complex (CPAC) and poly(A) binding proteins (PABPs). In the budding yeast Saccharomyces cerevisiae, the responsible PABP is the evolutionarily conserved CCCH zinc finger protein Nab2 that facilitates the biogenesis of ∼60 adenosine mRNA poly(A) tails. Here, we address the molecular basis for such length control. Reconstituting polyadenylation reactions during the formation of Nab2:poly(A) RNA ribonucleoprotein particles in vitro, we found that Nab2 dimerization directs polyadenylation termination. The Nab2 dimer is stable only on poly(A) tails that are >25 adenosines, explaining how Nab2 avoids prematurely terminating poly(A) synthesis. However, the mature tail length is not determined by the footprint of Nab2 on the RNA but rather by the kinetic competition between CPAC-mediated tail elongation and Nab2 RNA binding. Variations in Nab2 RNA binding rate can shift poly(A) tail lengths, but in cells such variations are buffered by autoregulation of Nab2 protein concentration. As a result, poly(A) tail length control operates through a "kinetic ruler" mechanism, whereby the concentration of Nab2 quantifies RNA length.

Shortening of messenger RNA poly(A) tails by the Ccr4-Not complex initiates mRNA decay and is a major determinant of gene regulation. RNA adaptors modulate the specificity of deadenylation by binding to Ccr4-Not through their intrinsically disordered regions (IDRs). However, the determinants of specificity and their regulation are largely unclear. Here we use nuclear magnetic resonance spectroscopy, biochemical reconstitution and structural modeling to show that dispersed segments within the IDR of the fission yeast Puf3 RNA adaptor interact with Ccr4-Not, consistent with multivalency. Binding can be modulated by phosphorylation, altering the deadenylation rate in a continuously tunable manner. Regulation of deadenylation through multivalency and phosphorylation likely occurs in evolutionarily divergent IDRs from additional RNA adaptors, including human Pumilio and Tristetraprolin. Overall, our in vitro data suggest that mRNA decay can be regulated not only as a bistable on-off switch but also by a graded mechanism, rationalizing how post-transcriptional gene expression can be fine-tuned.

mRNA decay is a major determinant of gene regulation that is controlled through shortening of mRNA poly(A) tails by the Ccr4-Not complex. The specificity of deadenylation can be mediated through RNA adaptors - RNA-binding proteins that tether substrate mRNAs to Ccr4-Not in a regulated and context-specific manner. Interaction with Ccr4-Not is mediated by intrinsically disordered regions (IDRs) within the RNA adaptors. Due to the difficulty in studying large IDR-containing complexes, the determinants of specificity and their regulation remain unclear. Here we use structural biology and biochemical reconstitution to show that dispersed segments within IDRs of RNA adaptors bind to several distinct binding sites on Ccr4-Not through multivalent interactions. We further demonstrate that binding can be modulated by phosphorylation, altering the consequent deadenylation rate in a continuously tunable manner. This mechanism is broadly applicable in evolutionarily divergent IDRs from multiple RNA adaptors including fission yeast Puf3, and human Pumilio/PUM1 and Tristetraprolin/TTP. Together, our work suggests that multivalent interactions and phosphorylation represent conserved strategies for regulating gene expression. Thus, in response to cellular cues, mRNA decay can be regulated by a graded mechanism, rather than a bistable on/off switch, rationalizing how post-transcriptional gene expression is fine-tuned. ### Competing Interest Statement The authors have declared no competing interest.

The interconnections between co-transcriptional regulation, chromatin environment and transcriptional output remain poorly understood. Here, we investigate the mechanism underlying RNA 3 processing-mediated Polycomb silencing of Arabidopsis FLOWERING LOCUS C (FLC). We show a requirement for APRF1, a homologue of yeast Swd2 and human WDR82, known to regulate RNA Pol II during transcription termination. APRF1 interacts with TOPP4 (yeast Glc7/human PP1) and LD, the latter showing structural features found in Ref2/PNUTS; all components of the yeast and human phosphatase module of the CPF 3 end processing machinery. LD has been shown to co-associate in vivo with the histone H3 K4 demethylase FLD. We show APRF1 and LD couple CPF-mediated cleavage and polyadenylation with removal of H3K4 monomethylation in the body of FLC, and this influences subsequent transcription. This work shows how transcription termination can change the local chromatin environment to modulate transcription of Arabidopsis FLC and affect flowering time.

To celebrate the 50th anniversary of Cell Press and the Cell focus issue on structural biology, we discussed with scientists working across diverse fields how AlphaFold has changed their research and brought structural biology to the masses.

Since Nature Structural and Molecular Biology was started 30 years ago, our understanding of transcription and mRNA processing has been revolutionized through structural and mechanistic studies. Here, we present our personal views of the advances in understanding the production of mature eukaryotic mRNAs over the past decade.

DNA crosslinks block DNA replication and are repaired by the Fanconi anaemia pathway. The FANCD2-FANCI (D2-I) protein complex is central to this process as it initiates repair by coordinating DNA incisions around the lesion1. However, D2-I is also known to have a more general role in DNA repair and in protecting stalled replication forks from unscheduled degradation2-4. At present, it is unclear how DNA crosslinks are recognized and how D2-I functions in replication fork protection. Here, using single-molecule imaging, we show that D2-I is a sliding clamp that binds to and diffuses on double-stranded DNA. Notably, sliding D2-I stalls on encountering single-stranded-double-stranded (ss-ds) DNA junctions, structures that are generated when replication forks stall at DNA lesions5. Using cryogenic electron microscopy, we determined structures of D2-I on DNA that show that stalled D2-I makes specific interactions with the ss-dsDNA junction that are distinct from those made by sliding D2-I. Thus, D2-I surveys dsDNA and, when it reaches an ssDNA gap, it specifically clamps onto ss-dsDNA junctions. Because ss-dsDNA junctions are found at stalled replication forks, D2-I can identify sites of DNA damage. Therefore, our data provide a unified molecular mechanism that reconciles the roles of D2-I in the recognition and protection of stalled replication forks in several DNA repair pathways.

Microtubules play crucial roles in cellular architecture, intracellular transport, and mitosis. The availability of free tubulin subunits affects polymerization dynamics and microtubule function. When cells sense excess free tubulin, they trigger degradation of the encoding mRNAs, which requires recognition of the nascent polypeptide by the tubulin-specific ribosome-binding factor TTC5. How TTC5 initiates the decay of tubulin mRNAs is unknown. Here, our biochemical and structural analysis reveals that TTC5 recruits the poorly studied protein SCAPER to the ribosome. SCAPER, in turn, engages the CCR4-NOT deadenylase complex through its CNOT11 subunit to trigger tubulin mRNA decay. SCAPER mutants that cause intellectual disability and retinitis pigmentosa in humans are impaired in CCR4-NOT recruitment, tubulin mRNA degradation, and microtubule-dependent chromosome segregation. Our findings demonstrate how recognition of a nascent polypeptide on the ribosome is physically linked to mRNA decay factors via a relay of protein-protein interactions, providing a paradigm for specificity in cytoplasmic gene regulation.

Lori Passmore is a Group Leader at the MRC Laboratory of Molecular Biology (MRC‐LMB). She studied Biochemistry at the University of British Columbia in Vancouver (Canada), before moving to the UK in 1999 for a PhD at the Institute of Cancer Research. After completing her PhD, Lori moved to Cambridge, where she became a Post‐Doctoral Fellow at the MRC‐LMB. In 2009, Lori started her own group at the MRC‐LMB and was subsequently awarded an ERC Starting Grant (2011), an ERC Consolidator Grant (2017) and a Wellcome Discovery Award (2023). She was also elected into the EMBO Young Investigator Programme (2015) and EMBO Membership (2018). Lori's research focusses on the determination of the structures of protein complexes that regulate gene expression, using primarily cryo‐electron microscopy and in vitro assays. Her work has contributed significantly to our understanding of the underlying molecular mechanisms of cellular processes, giving insights into human physiology and disease. In this interview, Lori provides an overview of her research and discusses current challenges in the field, recalls the key events and collaborations that have helped shape her successful research career and offers advice to early career scientists.

Gene expression is controlled in a dynamic and regulated manner to allow for the consistent and steady expression of some proteins as well as the rapidly changing production of other proteins. Transcription initi-ation has been a major focus of study because it is highly regulated. However, termination of transcription also plays an important role in controlling gene expression. Transcription termination on protein-coding genes is intimately linked with 3' end cleavage and polyadenylation of transcripts, and it generally results in the production of a mature mRNA that is exported from the nucleus. Termination on many non-coding genes can also result in the production of a mature transcript. Termination is dynamically regulated-prema-ture termination and transcription readthrough occur in response to a number of cellular signals, and these can have varied consequences on gene expression. Here, we review eukaryotic transcription termination by RNA polymerase II (RNAPII), focusing on protein-coding genes.

In vitro reconstitution of recognition of 80S ribosomes by CCR4-NOT, cryo-electron microscopy (cryoEM), crosslinking mass spectrometry and biochemical characterization reveal that CCR4-NOT specifically identifies ribosomes stalled during translation elongation. CCR4-NOT occupies the ribosomal exit site (E site) and locks the ribosomal L1 stalk in an open conformation to enforce the stalled state.

Formation of the 3′ end of a eukaryotic mRNA is a key step in the production of a mature transcript. This process is mediated by a number of protein factors that cleave the pre-mRNA, add a poly(A) tail, and regulate transcription by protein dephosphorylation. Cleavage and polyadenylation specificity factor (CPSF) in humans, or cleavage and polyadenylation factor (CPF) in yeast, coordinates these enzymatic activities with each other, with RNA recognition, and with transcription. The site of pre-mRNA cleavage can strongly influence the translation, stability, and localization of the mRNA. Hence, cleavage site selection is highly regulated. The length of the poly(A) tail is also controlled to ensure that every transcript has a similar tail when it is exported from the nucleus. In this review, we summarize new mechanistic insights into mRNA 3′-end processing obtained through structural studies and biochemical reconstitution and outline outstanding questions in the field.

Translation affects messenger RNA stability and, in yeast, this is mediated by the Ccr4-Not deadenylation complex. The details of this process in mammals remain unclear. Here, we use cryogenic electron microscopy (cryo-EM) and crosslinking mass spectrometry to show that mammalian CCR4-NOT specifically recognizes ribosomes that are stalled during translation elongation in an in vitro reconstituted system with rabbit and human components. Similar to yeast, mammalian CCR4-NOT inserts a helical bundle of its CNOT3 subunit into the empty E site of the ribosome. Our cryo-EM structure shows that CNOT3 also locks the L1 stalk in an open conformation to inhibit further translation. CCR4-NOT is required for stable association of the nonconstitutive subunit CNOT4, which ubiquitinates the ribosome, likely to signal stalled translation elongation. Overall, our work shows that human CCR4-NOT not only detects but also enforces ribosomal stalling to couple translation and mRNA decay.

Transcription termination by RNA Polymerase II (Pol II) is linked to RNA 3ʹ-end processing by the cleavage and polyadenylation factor (CPF). CPF contains endonuclease, poly(A) polymerase and protein phosphatase activities that cleave and polyadenylate the pre-mRNA, and dephosphorylate Pol II to control transcription. Exactly how the RNA 3ʹ-end processing machinery is coupled to transcription remains unclear. Here, we combine in vitro reconstitution, electron cryo-microscopy and X-ray crystallography to show that CPF physically and functionally interacts with Pol II. Surprisingly, CPF-mediated dephosphorylation promotes the formation of a Pol II stalk-to-stalk homodimer. This dimer is compatible with transcription (unlike other Pol II dimers) but not with the binding of transcription elongation factors. We show that the Ref2 subunit of CPF contributes directly to the interaction with Pol II and also regulates the Glc7/PP1 CPF phosphatase. We hypothesize that Pol II dimerization may play a role in gene looping and may provide a mechanistic basis for the allosteric model of transcription termination.

3’-end processing of most human mRNAs is carried out by the cleavage and polyadenylation specificity factor (CPSF; CPF in yeast). Endonucleolytic cleavage of the nascent pre-mRNA defines the 3’-end of the mature transcript, which is important for mRNA localization, translation and stability. Cleavage must therefore be tightly regulated. Here, we reconstitute specific and efficient 3’-endonuclease activity of human CPSF with purified proteins. This requires the sevensubunit CPSF as well as three additional protein factors: cleavage stimulatory factor (CStF), cleavage factor IIm (CFIIm) and, importantly, the multi-domain protein RBBP6. Unlike its yeast homologue Mpe1, which is a stable subunit of CPF, RBBP6 does not copurify with CPSF and is recruited in an RNA-dependent manner. Sequence and mutational analyses suggest that RBBP6 interacts with the WDR33 and CPSF73 subunits of CPSF. Thus, it is likely that the role of RBBP6 is conserved from yeast to human. Overall, our data are consistent with CPSF endonuclease activation and site-specific pre-mRNA cleavage being highly controlled to maintain fidelity in RNA processing.