scholarly journals Three-layered control of mRNA poly(A) tail synthesis in Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Matti Turtola ◽  
M. Cemre Manav ◽  
Ananthanarayanan Kumar ◽  
Agnieszka Tudek ◽  
Seweryn Mroczek ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Mrna Export ◽  
Tail Length ◽  
Control Mechanisms ◽  
Eukaryotic Mrnas ◽  
Cleavage And Polyadenylation ◽  
Fail Safe ◽  
Dependent Pathway ◽  
Polyadenylation Factor

Biogenesis of most eukaryotic mRNAs involves the addition of an untemplated polyadenosine (pA) tail by the cleavage and polyadenylation machinery. The pA tail, and its exact length, impacts mRNA stability, nuclear export, and translation. To define how polyadenylation is controlled in S. cerevisiae, we have used an in vivo assay capable of assessing nuclear pA tail synthesis, analyzed tail length distributions by direct RNA sequencing, and reconstituted polyadenylation reactions with purified components. This revealed three control mechanisms for pA tail length. First, we found that the pA binding protein (PABP) Nab2p is the primary regulator of pA tail length. Second, when Nab2p is limiting, the nuclear pool of Pab1p, the second major PABP in yeast, controls the process. Third, when both PABPs are absent, the cleavage and polyadenylation factor (CPF) limits pA tail synthesis. Thus, Pab1p and CPF provide fail-safe mechanisms to a primary Nab2p-dependent pathway, thereby preventing uncontrolled polyadenylation and allowing mRNA export and translation.

scholarly journals Yeast Pab1 interacts with Rna15 and participates in the control of the poly(A) tail length in vitro.

1997 ◽  
Vol 17 (7) ◽  
pp. 3694-3701 ◽  
Author(s):  
N Amrani ◽  
M Minet ◽  
M Le Gouar ◽  
F Lacroute ◽  
F Wyers
Keyword(s):  
Wild Type Strain ◽  
Tail Length ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Wild Type ◽  
Cleavage And Polyadenylation ◽  
Two Hybrid ◽  
Polyadenylation Factor

In Saccharomyces cerevisiae, the single poly(A) binding protein, Pab1, is the major ribonucleoprotein associated with the poly(A) tails of mRNAs in both the nucleus and the cytoplasm. We found that Pab1 interacts with Rna15 in two-hybrid assays and in coimmunoprecipitation experiments. Overexpression of PAB1 partially but specifically suppressed the rna15-2 mutation in vivo. RNA15 codes for a component of the cleavage and polyadenylation factor CF I, one of the four factors needed for pre-mRNA 3'-end processing. We show that Pab1 and CF I copurify in anion-exchange chromatography. These data suggest that Pab1 is physically associated with CF I. Extracts from a thermosensitive pab1 mutant and from a wild-type strain immunoneutralized for Pab1 showed normal cleavage activity but a large increase in poly(A) tail length. A normal tail length was restored by adding recombinant Pab1 to the mutant extract. The longer poly(A) tails were not due to an inhibition of exonuclease activities. Pab1 has previously been implicated in the regulation of translation initiation and in cytoplasmic mRNA stability. Our data indicate that Pab1 is also a part of the 3'-end RNA-processing complex and thus participates in the control of the poly(A) tail lengths during the polyadenylation reaction.

scholarly journals Coordination of Hpr1 and Ubiquitin Binding by the UBA Domain of the mRNA Export Factor Mex67

2007 ◽  
Vol 18 (7) ◽  
pp. 2561-2568 ◽  
Author(s):  
Maria Hobeika ◽  
Christoph Brockmann ◽  
Nahid Iglesias ◽  
Carole Gwizdek ◽  
David Neuhaus ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Conformational Change ◽  
Nuclear Export ◽  
Transcription Elongation ◽  
Mrna Export ◽  
Molecular Switch ◽  
Ubiquitin Binding ◽  
Uba Domain ◽  
Nuclear Export Receptor

The ubiquitin-associated (UBA) domain of the mRNA nuclear export receptor Mex67 helps in coordinating transcription elongation and nuclear export by interacting both with ubiquitin conjugates and specific targets, such as Hpr1, a component of the THO complex. Here, we analyzed substrate specificity and ubiquitin selectivity of the Mex67 UBA domain. UBA-Mex67 is formed by three helices arranged in a classical UBA fold plus a fourth helix, H4. Deletion or mutation of helix H4 strengthens the interaction between UBA-Mex67 and ubiquitin, but it decreases its affinity for Hpr1. Interaction with Hpr1 is required for Mex67 UBA domain to bind polyubiquitin, possibly by inducing an H4-dependent conformational change. In vivo, deletion of helix H4 reduces cotranscriptional recruitment of Mex67 on activated genes, and it also shows an mRNA export defect. Based on these results, we propose that H4 functions as a molecular switch that coordinates the interaction of Mex67 with ubiquitin bound to specific substrates, defines the selectivity of the Mex67 UBA domain for polyubiquitin, and prevents its binding to nonspecific substrates.

Kinetics of mRNA polyadenylation and its relation to transcription termination in eukaryotes

2000 ◽  
Vol 28 (5) ◽  
pp. A461-A461
Author(s):  
Amer Jamil ◽  
Harold G. Martinson
Keyword(s):  
Transcription Termination ◽  
Rnase Protection Assay ◽  
Nascent Transcript ◽  
Rnase Protection ◽  
Multistep Process ◽  
Eukaryotic Mrnas ◽  
Cleavage And Polyadenylation ◽  
A Site ◽  
Antisense Element

Processing of most eukaryotic mRNAs includes and polyadenylation of the nascent transcript. Until now there has been no method for the reliable measurement of these processes in vivo. Therefore, in the present work a new technique was developed for measuring precisely the rate of cleavage and polyadenylation in vivo. The method uses a cis-antisense element targeted to an upstream poly (A) signal. Duplex formation of the antisense element with the poly (A) signal region prevents polyadenylation. In a series of expression vector constructs the antisense element was moved increasing distances downstream of its target poly (A) site, reasoning that if it takes the polymerase longer to reach the antisense element, polyadenylation would have more time to occur. Using this method the half time for commitment to cleavage and polyadenylation at the SV40 early poly (A) site and SPA (synthetic poly A site) was found to be 5 seconds. It was found that strong sites (SV 40 late poly (A) site) were processed faster. Commitment to cleavage and polyadenylation was found to be a multistep process. The expression results were confirmed with the help of RNase protection assay. Relationship between polyadenylation and transcription termination was also studied by using G-free assay and found to be positively correlated. Present data support looping moded suggesting some communication exists between polyadenylation complex and RNA pol. II for transcription termination.

scholarly journals Analysis of Dom34 and Its Function in No-Go Decay

2009 ◽  
Vol 20 (13) ◽  
pp. 3025-3032 ◽  
Author(s):  
Dario O. Passos ◽  
Meenakshi K. Doma ◽  
Christopher J. Shoemaker ◽  
Denise Muhlrad ◽  
Rachel Green ◽  
...  
Keyword(s):  
Translation Termination ◽  
Protein A ◽  
Cold Sensitivity ◽  
Control Mechanisms ◽  
Functional Regions ◽  
Endonucleolytic Cleavage ◽  
Mrna Cleavage ◽  
Eukaryotic Mrnas ◽  
Multistep Model

Eukaryotic mRNAs are subject to quality control mechanisms that degrade defective mRNAs. In yeast, mRNAs with stalls in translation elongation are targeted for endonucleolytic cleavage by No-Go decay (NGD). The cleavage triggered by No-Go decay is dependent on Dom34p and Hbs1p, and Dom34 has been proposed to be the endonuclease responsible for mRNA cleavage. We created several Dom34 mutants and examined their effects on NGD in yeast. We identified mutations in several loops of the Dom34 structure that affect NGD. In contrast, mutations inactivating the proposed nuclease domain do not affect NGD in vivo. Moreover, we observed that overexpression of the Rps30a protein, a high copy suppressor of dom34Δ cold sensitivity, can restore some mRNA cleavage in a dom34Δ strain. These results identify important functional regions of Dom34 and suggest that the proposed endonuclease activity of Dom34 is not required for mRNA cleavage in NGD. We also provide evidence that the process of NGD is conserved in insect cells. On the basis of these results and the process of translation termination, we suggest a multistep model for the process of NGD.

scholarly journals Crp79p, Like Mex67p, Is an Auxiliary mRNA Export Factor inSchizosaccharomyces pombe

2002 ◽  
Vol 13 (8) ◽  
pp. 2571-2584 ◽  
Author(s):  
Anjan G. Thakurta ◽  
William A. Whalen ◽  
Jin Ho Yoon ◽  
Anekella Bharathi ◽  
Libor Kozak ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Mrna Export ◽  
Nuclear Pore ◽  
Rna Recognition Motif ◽  
Synthetic Lethal ◽  
Export Activity ◽  
C Terminus ◽  
Green Fluorescent

The export of mRNA from the nucleus to the cytoplasm involves interactions of proteins with mRNA and the nuclear pore complex. We isolated Crp79p, a novel mRNA export factor from the same synthetic lethal screen that led to the identification of spMex67p inSchizosaccharomyces pombe. Crp79p is a 710-amino-acid-long protein that contains three RNA recognition motif domains in tandem and a distinct C-terminus. Fused to green fluorescent protein (GFP), Crp79p localizes to the cytoplasm. Like Mex67p, Crp79-GFP binds poly(A)+ RNA in vivo, shuttles between the nucleus and the cytoplasm, and contains a nuclear export activity at the C-terminus that is Crm1p-independent. All of these properties are essential for Crp79p to promote mRNA export. Crp79p import into the nucleus depends on the Ran system. A domain of spMex67p previously identified as having a nuclear export activity can functionally substitute for the nuclear export activity at the C-terminus of Crp79p. Although both Crp79p and spMex67p function to export mRNA, Crp79p does not substitute for all of spMex67p functions and probably is not a functional homologue of spMex67p. We propose that Crp79p is a nonessential mRNA export carrier in S. pombe.

scholarly journals Assembly of an Export-Competent mRNP Is Needed for Efficient Release of the 3′-End Processing Complex after Polyadenylation

2009 ◽  
Vol 29 (19) ◽  
pp. 5327-5338 ◽  
Author(s):  
Xiangping Qu ◽  
Søren Lykke-Andersen ◽  
Tommy Nasser ◽  
Cyril Saguez ◽  
Edouard Bertrand ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Growth Defect ◽  
Nuclear Pore ◽  
Rna Immunoprecipitation ◽  
Cleavage And Polyadenylation ◽  
Severe Growth Defect ◽  
Nuclear Export Receptor ◽  
Polyadenylated Mrna

ABSTRACT Before polyadenylated mRNA is exported from the nucleus, the 3′-end processing complex is removed by a poorly described mechanism. In this study, we asked whether factors involved in mRNP maturation and export are also required for disassembly of the cleavage and polyadenylation complex. An RNA immunoprecipitation assay monitoring the amount of the cleavage factor (CF) IA component Rna15p associated with poly(A)+ RNA reveals defective removal of Rna15p in mutants of the nuclear export receptor Mex67p as well as other factors important for assembly of an export-competent mRNP. In contrast, Rna15p is not retained in mutants of export factors that function primarily on the cytoplasmic side of the nuclear pore. Consistent with a functional interaction between Mex67p and the 3′-end processing complex, a mex67 mutant accumulates unprocessed SSA4 transcripts and exhibits a severe growth defect when this mutation is combined with mutation of Rna15p or another CF IA subunit, Rna14p. RNAs that become processed in a mex67 mutant have longer poly(A) tails both in vivo and in vitro. This influence of Mex67p on 3′-end processing is conserved, as depletion of its human homolog, TAP/NXF1, triggers mRNA hyperadenylation. Our results indicate a function for nuclear mRNP assembly factors in releasing the 3′-end processing complex once polyadenylation is complete.

Intron excision from precursor tRNA molecules in mammalian cells requires ATP hydrolysis and phosphorylation of tRNA-splicing endonuclease components

2013 ◽  
Vol 41 (4) ◽  
pp. 831-837 ◽  
Author(s):  
Barbara Mair ◽  
Johannes Popow ◽  
Karl Mechtler ◽  
Stefan Weitzer ◽  
Javier Martinez
Keyword(s):  
Mammalian Cells ◽  
Kinase Activity ◽  
Atp Hydrolysis ◽  
Generate Functional ◽  
Trna Splicing ◽  
Cleavage And Polyadenylation ◽  
Motor Neuron Loss ◽  
Polyadenylation Factor

The process of tRNA splicing entails removal of an intron by TSEN (tRNA–splicing endonuclease) and ligation of the resulting exon halves to generate functional tRNAs. In mammalian cells, the RNA kinase CLP1 (cleavage and polyadenylation factor I subunit) associates with TSEN and phosphorylates the 3′ exon at the 5′ end in vitro, suggesting a role for CLP1 in tRNA splicing. Interestingly, recent data suggest that the ATP-binding and/or hydrolysis capacity of CLP1 is required to enhance pre-tRNA cleavage. In vivo, the lack of CLP1 kinase activity leads to progressive motor neuron loss and accumulation of novel 5′ leader–5′ exon tRNA fragments. We have extended the investigation of the biochemical requirements in pre-tRNA splicing and found that β–γ-hydrolysable ATP is crucial for the productive generation of exon halves. In addition, we provide evidence that phosphorylation of the TSEN complex components supports efficient pre-tRNA cleavage. Taken together, our data improve the mechanistic understanding of mammalian pre-tRNA processing and its regulation.

scholarly journals Yeast Ran-Binding Protein 1 (Yrb1) Shuttles between the Nucleus and Cytoplasm and Is Exported from the Nucleus via a CRM1(XPO1)-Dependent Pathway

2000 ◽  
Vol 20 (12) ◽  
pp. 4295-4308 ◽  
Author(s):  
Markus Künzler ◽  
Thomas Gerstberger ◽  
Françoise Stutz ◽  
F. Ralf Bischoff ◽  
Ed Hurt
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Binding Protein ◽  
Nuclear Pore ◽  
Leptomycin B ◽  
Import And Export ◽  
Yeast Homolog ◽  
Dependent Pathway ◽  
Cytoplasmic Side

ABSTRACT The RanGTP-binding protein RanBP1, which is located in the cytoplasm, has been implicated in release of nuclear export complexes from the cytoplasmic side of the nuclear pore complex. Here we show that Yrb1 (the yeast homolog of RanBP1) shuttles between the nucleus and the cytoplasm. Nuclear import of Yrb1 is a facilitated process that requires a short basic sequence within the Ran-binding domain (RBD). By contrast, nuclear export of Yrb1 requires an intact RBD, which forms a ternary complex with the Xpo1 (Crm1) NES receptor in the presence of RanGTP. Nuclear export of Yrb1, however, is insensitive towards leptomycin B, suggesting a novel type of substrate recognition between Yrb1 and Xpo1. Taken together, these data suggest that ongoing nuclear import and export is an important feature of Yrb1 function in vivo.

scholarly journals A U2-snRNP–independent role of SF3b in promoting mRNA export

2019 ◽  
Vol 116 (16) ◽  
pp. 7837-7846 ◽  
Author(s):  
Ke Wang ◽  
Changping Yin ◽  
Xian Du ◽  
Suli Chen ◽  
Jianshu Wang ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Mrna Export ◽  
Mrna Processing ◽  
Dependent Manner ◽  
U2 Snrnp ◽  
Dose Dependent ◽  
Antisense Morpholino

To ensure efficient and accurate gene expression, pre-mRNA processing and mRNA export need to be balanced. However, how this balance is ensured remains largely unclear. Here, we found that SF3b, a component of U2 snRNP that participates in splicing and 3′ processing of pre-mRNAs, interacts with the key mRNA export adaptor THO in vivo and in vitro. Depletion of SF3b reduces THO binding with the mRNA and causes nuclear mRNA retention. Consistently, introducing SF3b binding sites into the mRNA enhances THO recruitment and nuclear export in a dose-dependent manner. These data demonstrate a role of SF3b in promoting mRNA export. In support of this role, SF3b binds with mature mRNAs in the cells. Intriguingly, disruption of U2 snRNP by using a U2 antisense morpholino oligonucleotide does not inhibit, but promotes, the role of SF3b in mRNA export as a result of enhanced SF3b–THO interaction and THO recruitment to the mRNA. Together, our study uncovers a U2-snRNP–independent role of SF3b in mRNA export and suggests that SF3b contributes to balancing pre-mRNA processing and mRNA export.

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

2021 ◽  
Author(s):  
Juan B Rodriguez-Molina ◽  
Francis J O'Reilly ◽  
Eleanor Sheekey ◽  
Sarah Maslen ◽  
J Mark Skehel ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Signal Sequence ◽  
Transcription Termination ◽  
Polyadenylation Signal ◽  
Messenger Rnas ◽  
Endonucleolytic Cleavage ◽  
Cleavage And Polyadenylation ◽  
Polyadenylation Factor ◽  
Transcription Interference

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. 

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