Architecture of eukaryotic mRNA 3'-end processing machinery.
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ABSTRACT:
SUBMITTER: Casanal A
PROVIDER: S-EPMC5788269 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
| S-EPMC7614891 | biostudies-literature
Project description: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 labeled Fip1 into recombinant CPF, we could study the dynamics of Fip1 within the megadalton complex using nuclear magnetic resonance (NMR) spectroscopy. 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 within the 3' end processing machinery are required to coordinate cleavage and polyadenylation.
| S-EPMC8559680 | biostudies-literature
Project description:Messenger RNA (mRNA) 3' end formation is a nuclear process through which all eukaryotic primary transcripts are endonucleolytically cleaved and most of them acquire a poly(A) tail. This process, which consists in the recognition of defined poly(A) signals of the pre-mRNAs by a large cleavage/polyadenylation machinery, plays a critical role in gene expression. Indeed, the poly(A) tail of a mature mRNA is essential for its functions, including stability, translocation to the cytoplasm and translation. In addition, this process serves as a bridge in the network connecting the different transcription, capping, splicing and export machineries. It also participates in the quantitative and qualitative regulation of gene expression in a variety of biological processes through the selection of single or alternative poly(A) signals in transcription units. A large number of protein factors associates with this machinery to regulate the efficiency and specificity of this process and to mediate its interaction with other nuclear events. Here, we review the eukaryotic 3' end processing machineries as well as the comprehensive set of regulatory factors and discuss the different molecular mechanisms of 3' end processing regulation by proposing several overlapping models of regulation.
| S-EPMC2874999 | biostudies-literature
Project description: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 reconstituted specific and efficient 3' endonuclease activity of human CPSF with purified proteins. This required the seven-subunit CPSF as well as three additional protein factors: cleavage stimulatory factor (CStF), cleavage factor IIm (CFIIm), and, importantly, the multidomain protein RBBP6. Unlike its yeast homolog 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 humans. Overall, our data are consistent with CPSF endonuclease activation and site-specific pre-mRNA cleavage being highly controlled to maintain fidelity in mRNA processing.
| S-EPMC8887125 | biostudies-literature
Project description:The mammalian pre-mRNA 3'-end-processing machinery consists of cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), and other proteins, but the overall architecture of this machinery remains unclear. CPSF contains two functionally distinct modules: a cleavage factor (mCF) and a polyadenylation specificity factor (mPSF). Here, we have produced recombinant human CPSF and CstF and examined these factors by electron microscopy (EM). We find that mPSF is the organizational core of the machinery, while the conformations of mCF and CstF and the position of mCF relative to mPSF are highly variable. We have identified by cryo-EM a segment in CPSF100 that tethers mCF to mPSF, and we have named it the PSF interaction motif (PIM). Mutations in the PIM can abolish CPSF formation, indicating that it is a crucial contact in CPSF. We have also obtained reconstructions of mCF and CstF77 by cryo-EM, assembled around the mPSF core.
| S-EPMC7036032 | biostudies-literature
Project description:The 3'-end processing machinery for metazoan replication-dependent histone precursor messenger RNAs (pre-mRNAs) contains the U7 small nuclear ribonucleoprotein and shares the key cleavage module with the canonical cleavage and polyadenylation machinery. We reconstituted an active human histone pre-mRNA processing machinery using 13 recombinant proteins and two RNAs and determined its structure by cryo-electron microscopy. The overall structure is highly asymmetrical and resembles an amphora with one long handle. We captured the pre-mRNA in the active site of the endonuclease, the 73-kilodalton subunit of the cleavage and polyadenylation specificity factor, poised for cleavage. The endonuclease and the entire cleavage module undergo extensive rearrangements for activation, triggered through the recognition of the duplex between the authentic pre-mRNA and U7 small nuclear RNA (snRNA). Our study also has notable implications for understanding canonical and snRNA 3'-end processing.
| S-EPMC7008720 | biostudies-literature
Project description:Processing of mRNA precursors (pre-mRNAs) by polyadenylation is an essential step in gene expression. Polyadenylation consists of two steps, cleavage and poly(A) synthesis, and requires multiple cis elements in the pre-mRNA and a megadalton protein complex bearing the two essential enzymatic activities. While genetic and biochemical studies remain the major approaches in characterizing these factors, structural biology has emerged during the past decade to help understand the molecular assembly and mechanistic details of the process. With structural information about more proteins and higher-order complexes becoming available, we are coming closer to obtaining a structural blueprint of the polyadenylation machinery that explains both how this complex functions and how it is regulated and connected to other cellular processes.
| S-EPMC4019069 | biostudies-literature
Project description:Firczuk2013 - Eukaryotic mRNA translation machinery
This is a model of Saccharomyces cerevisiae
mRNA translation which includes the initiation, elongation and termination phases. The model is for 20 condon mRNAs. The building of a multi-factor complex in initiation and also the different processes in elongation and termination are modelled in detail. The model takes into account that ribosomes cover more than one codon of mRNA so that the movement of ribosomes are effectively blocked by other ribosomes several codons downstream. It is assumed that 15 codons are occupied by each ribosome. This blocking effect is considered in reaction R18 in initiation and also reaction R26, the reaction where translocation of ribosomes takes place in elongation. The kinetic functions of these two reactions are based on MacDonald et al. 1968 and Heinrich & Rapaport 1980. All other kinetic functions follow mass-action kinetics. The concentrations of transfer RNA species (Met-tRNA, aa-tRNA and tRNA in the model) are kept constant, while the other species' concentrations can change in the course of the simulation. The model describes the translation of a short mRNA with 20 codons. Therefore, all reactions in the elongation cycle (R22, R23, R25, R26, R28 and R29) and the corresponding species are replicated accordingly to model the species with ribosomes bound at different positions. In summary, the model contains 165 different species and 141 reactions.
The value of the 56 rate constant parameters were estimated by fitting the model against a series of experimental data consisting of modulation of the various translation factors (Figures 2, 3 and S3). Overall the parameter estimation was carried out over 212 different data points (steady states).
This model is described in the article:
An in vivo control map for the eukaryotic mRNA translation machinery
Helena Firczuk, Shichina Kannambath, Jürgen Pahle, Amy Claydon, Robert Beynon, John Duncan, Hans Westerhoff, Pedro Mendes and John EG McCarthy
Molecular Systems Biology. 9:635
Abstract:
Rate control analysis defines the in vivo control map governing yeast protein synthesis and generates an extensively parameterized digital model of the translation pathway. Among other non-intuitive outcomes, translation demonstrates a high degree of functional modularity and comprises a non-stoichiometric combination of proteins manifesting functional convergence on a shared maximal translation rate. In exponentially growing cells, polypeptide elongation (eEF1A, eEF2, and eEF3) exerts the strongest control. The two other strong control points are recruitment of mRNA and tRNAi to the 40S ribosomal subunit (eIF4F and eIF2) and termination (eRF1; Dbp5). In contrast, factors that are found to promote mRNA scanning efficiency on a longer than-average 5′untranslated region (eIF1, eIF1A, Ded1, eIF2B, eIF3, and eIF5) exceed the levels required for maximal control. This is expected to allow the cell to minimize scanning transition times, particularly for longer 5′UTRs. The analysis reveals these and other collective adaptations of control shared across the factors, as well as features that reflect functional modularity and system robustness. Remarkably, gene duplication is implicated in the fine control of cellular protein synthesis.
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2024-09-02 | BIOMD0000000457 | BioModels
Project description:The polyadenosine (poly(A)) tail found on the 3'-end of almost all eukaryotic mRNAs is important for mRNA stability and regulation of translation. mRNA 3'-end processing occurs co-transcriptionally and involves more than 20 proteins to specifically recognize the polyadenylation site, cleave the pre-mRNA, add a poly(A) tail, and trigger transcription termination. The polyadenylation site (PAS) defines the end of the 3'-untranslated region (3'-UTR) and, therefore, selection of the cleavage site is a critical event in regulating gene expression. Integrated structural biology approaches including biochemical reconstitution of multi-subunit complexes, cross-linking mass spectrometry, and structural analyses by X- ray crystallography and single-particle electron cryo-microscopy (cryoEM) have enabled recent progress in understanding the molecular mechanisms of the mRNA 3'-end processing machinery. Here, we describe new molecular insights into pre-mRNA recognition, cleavage and polyadenylation.
| S-EPMC6900580 | biostudies-literature
Project description:Rate control analysis defines the in vivo control map governing yeast protein synthesis and generates an extensively parameterized digital model of the translation pathway. Among other non-intuitive outcomes, translation demonstrates a high degree of functional modularity and comprises a non-stoichiometric combination of proteins manifesting functional convergence on a shared maximal translation rate. In exponentially growing cells, polypeptide elongation (eEF1A, eEF2, and eEF3) exerts the strongest control. The two other strong control points are recruitment of mRNA and tRNA(i) to the 40S ribosomal subunit (eIF4F and eIF2) and termination (eRF1; Dbp5). In contrast, factors that are found to promote mRNA scanning efficiency on a longer than-average 5'untranslated region (eIF1, eIF1A, Ded1, eIF2B, eIF3, and eIF5) exceed the levels required for maximal control. This is expected to allow the cell to minimize scanning transition times, particularly for longer 5'UTRs. The analysis reveals these and other collective adaptations of control shared across the factors, as well as features that reflect functional modularity and system robustness. Remarkably, gene duplication is implicated in the fine control of cellular protein synthesis.
| S-EPMC3564266 | biostudies-other