Biology subjects

Passmore, L. A.

Publications and source records attributed to Passmore, L. A..

14 recordsLinked to original sources

bioRxiv2025

RNA-binding proteins provide specificity to the PAN2-PAN3 mRNA deadenylation complex

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.

molecular biology↗

bioRxiv2025

A kinetic ruler controls mRNA poly(A) tail length

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 find that Nab2 dimerization directs polyadenylation termination. The Nab2 dimer is stable only on poly(A) tails that are longer than 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.

molecular biology↗

bioRxiv2024

Phosphorylation-dependent tuning of mRNA deadenylation rates

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.

molecular biology↗

bioRxiv2024

Molecular insights into dynamic protein structures by high-contrast crosslinking mass spectrometry

Proteins are comprised of structured domains and dynamic regions, and both are essential for biological function. However, studying dynamic regions is challenging using most structural biology methods, including crosslinking mass spectrometry. Here, we dramatically improve the usefulness of distance restraints from crosslinking MS by taking advantage of short-lived reactive species generated from diazirine-based photo-crosslinking. This leads to a clear view of complex topologies and conformational changes, including in dynamic regions. We demonstrate that photo-crosslinking MS data can be used to model flexible regions and conformational changes in the DNA repair complexes; Fanconi Anemia core complex and FANCD2-FANCI. In addition, we obtain new insights into the architecture and arrangement of the highly flexible CCR4-NOT mRNA deadenylation complex. The improved contrast of photo-crosslinking empowers structural biology by providing clearer structural insights into dynamic biological systems that have eluded other structural biology approaches.

molecular biology↗

bioRxiv2023

The CPSF phosphatase module links transcription termination to chromatin silencing

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 3end 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.

molecular biology↗

bioRxiv2023

Mechanism of ribosome-associated mRNA degradation during tubulin autoregulation

SUMMARYMicrotubules play crucial roles in cellular architecture, intracellular transport, and mitosis. The availability of free tubulin subunits impacts 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 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.

molecular biology↗

bioRxiv2022

A direct interaction between CPF and Pol II links RNA 3'-end processing to transcription

Transcription termination by RNA Polymerase II (Pol II) is linked to RNA 3'-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 3'-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.

molecular biology↗

bioRxiv2022

Specific recognition and ubiquitination of slow-moving ribosomes by human CCR4-NOT

Eukaryotic messenger RNA (mRNA) decay is generally initiated by removal of the 3 polyadenosine (poly(A)) tail by the CCR4-NOT complex. Yeast Ccr4-Not binds and ubiquitinates ribosomes stalled on mRNAs with sub-optimal codons to trigger deadenylation and decay of the associated transcript. However, the mammalian ortholog of the E3 ubiquitin ligase subunit, CNOT4, is not a constitutive component of human CCR4-NOT. It therefore remains unclear how the mammalian deadenylation machinery targets stalled ribosomes. Here, we reconstitute translational stalling on non-optimal codons. We find that human CCR4-NOT recognizes translating mammalian ribosomes and is required for stable CNOT4 association. Our cryoEM structure reveals that the CNOT3 subunit detects slow translation and locks the L1 stalk of the ribosome in an open conformation to impede further elongation. Using crosslinking mass spectrometry, we show that CNOT4 and CNOT11 also bind in the vicinity of the E site. Overall, our work defines how CCR4-NOT enforces ribosomal stalling in response to low codon optimality.

molecular biology↗

bioRxiv2021

RBBP6 activates the pre-mRNA 3'-end processing machinery in humans

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.

molecular biology↗

bioRxiv2021

Mpe1 senses the polyadenylation signal in pre-mRNA to control cleavage and polyadenylation

Most eukaryotic messenger RNAs (mRNAs) are processed at their 3-end by the cleavage and polyadenylation factor (CPF/CPSF). CPF mediates endonucleolytic cleavage of the pre-mRNA and addition of a polyadenosine (poly(A)) tail, which together define the 3-end of the mature transcript. Activation of CPF is highly regulated to maintain fidelity of RNA processing. Here, using cryoEM of yeast CPF, we show that the Mpe1 subunit directly contacts the polyadenylation signal sequence in nascent pre- mRNA. This RNA-mediated link between the nuclease and polymerase modules promotes activation of the CPF endonuclease and controls polyadenylation. Mpe1 rearrangement is antagonized by another subunit, Cft2. In vivo, depletion of Mpe1 leads to widespread defects in transcription termination by RNA Polymerase II, resulting in transcription interference on neighboring genes. Together, our data suggest that Mpe1 plays a major role in selecting the cleavage site, activating CPF and ensuring timely transcription termination.

molecular biology↗

bioRxiv2021

Dynamics in Fip1 regulate eukaryotic mRNA 3'-end processing

Cleavage and polyadenylation factor (CPF/CPSF) is a multiprotein complex essential for mRNA 3-end processing in eukaryotes. It contains an endonuclease that cleaves pre-mRNAs, and a polymerase that adds a poly(A) tail onto the cleaved 3-end. Several CPF subunits, including Fip1, contain intrinsically-disordered regions (IDRs). IDRs within multiprotein complexes can be flexible, or can become ordered upon interaction with binding partners. Here, we show that yeast Fip1 anchors the poly(A) polymerase Pap1 onto CPF via an interaction with zinc finger 4 of another CPF subunit, Yth1. We also reconstitute a fully recombinant 850-kDa CPF. By incorporating selectively-labelled Fip1 into recombinant CPF, we could study the dynamics of this single protein within the megadalton complex using nuclear magnetic resonance spectroscopy (NMR). This reveals that a Fip1 IDR that connects the Yth1- and Pap1-binding sites remains highly dynamic within CPF. Together, our data suggest that Fip1 dynamics mediate conformational transitions within the 3-end processing machinery to coordinate cleavage and polyadenylation.

molecular biology↗

bioRxiv2020

Deep learning enables the atomic structure determination of the Fanconi Anemia core complex from cryoEM

Cryo-electron microscopy of protein complexes often leads to moderate resolution maps (4-8 [A]), with visible secondary structure elements but poorly resolved loops, making model-building challenging. In the absence of high-resolution structures of homologues, only coarse-grained structural features are typically inferred from these maps, and it is often impossible to assign specific regions of density to individual protein subunits. This paper describes a new method for overcoming these difficulties that integrates predicted residue distance distributions from a deep-learned convolutional neural network, computational protein folding using Rosetta, and automated EM-map-guided complex assembly. We apply this method to a 4.6 [A] resolution cryoEM map of Fanconi Anemia core complex (FAcc), an E3 ubiquitin ligase required for DNA interstrand crosslink repair, which was previously challenging to interpret as it is comprised of 6557 residues, only 1897 of which are covered by homology models. In the published structure built from this map, only 387 residues could be assigned to specific subunits. By building and placing into density 42 deep-learning guided models containing 4795 residues not included in the previously published structure, we are able to determine an almost-complete atomic model of FAcc, in which 5182 of the 6557 residues were placed. The resulting model is consistent with previously published biochemical data, and facilitates interpretation of disease related mutational data. We anticipate that our approach will be broadly useful for cryoEM structure determination of large complexes containing many subunits for which there are no homologues of known structure.

biochemistry↗

bioRxiv2019

FANCD2-FANCI is a clamp stabilized on DNA by monoubiquitination during DNA repair

Vertebrate DNA crosslink repair is a crucial process that excises toxic replication-blocking DNA interstrand crosslinks1. This pathway fails in the inherited human disease Fanconi anemia (FA), resulting in abnormal development, loss of blood production, and marked cancer susceptibility2. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause FA3. A biochemical description of how FA gene products might initiate the removal of DNA crosslinks is now emerging. However, structural insight into this vital process has been limited. Here, we use electron cryomicroscopy (cryoEM) to determine the structure of a key DNA crosslink repair factor - FANCD2 heterodimerized with FANCI (D2-I). Recombinant chicken D2-I adopts a conformation that is in agreement with the structure of its murine counter-part4. In contrast, the activated monoubiquitinated form of D2-I (ubD2-I) adopts an alternative closed conformation, creating a channel that encloses double-stranded DNA. Ubiquitin is positioned at the interface of FANCD2 and FANCI, and acts as a molecular pin to trap ubD2-I in the closed conformation, clamped on DNA. The new solvent-exposed interface around the monoubiquitination site is likely a platform for recruitment of other DNA repair factors. We also find that isolated FANCD2 is a dimer, but it adopts a closed conformation that is unable to bind DNA. When incubated with free FANCI, the FANCD2 homodimer exchanges to D2-I, acquiring DNA binding properties. This suggests an autoinhibitory mechanism that prevents FANCD2 activation on DNA until after assembly with FANCI. Together, our cryoEM and biochemical analysis suggests that D2-I is a clamp that is locked onto DNA by ubiquitin, and provides unanticipated new insight into the regulation and the initiation of DNA cross-link repair.

molecular biology↗

bioRxiv2018

The mechanism of Ccr4-Not recruitment to specific mRNAs involves sequence-selective tethering by RNA-binding proteins

The Ccr4-Not complex removes mRNA poly(A) tails to regulate eukaryotic mRNA stability and translation. RNA-binding proteins contribute to specificity but this is not fully understood. Here, we reconstitute accelerated and selective deadenylation of RNAs containing AU-rich elements (AREs) and Pumilio-response elements (PREs). We find that the fission yeast homologues of Tristetraprolin/TTP and Pumilio/Puf (Zfs1 and Puf3) act as molecular tethers: They recruit Ccr4-Not via multiple regions within low-complexity sequences, and bind specific RNA sequences via RNA-binding domains. Using a two-color assay to simultaneously monitor poly(A) tail removal from different RNAs, we demonstrate that Puf3 can distinguish between RNAs of very similar sequence. This is primarily due to differences in the dissociation rate constants. As a result, motif quality is a major determinant of mRNA stability for Puf3 targets in vivo. Together, we provide new insight into the selective deadenylation of specific mRNAs by Ccr4-Not, and the prediction of targeted mRNAs.

molecular biology↗