Molecular dissection of mRNA poly(A) tail length control in yeast

In eukaryotic cells, newly synthesized mRNAs acquire a poly(A) tail that plays several fundamental roles in export, translation and mRNA decay. In mammals, PABPN1 controls the processivity of polyadenylation and the length of poly(A) tails during de novo synthesis. This regulation is less well-detailed in yeast. We have recently demonstrated that Nab2p is necessary and sufficient for the regulation of polyadenylation and that the Pab1p/PAN complex may act at a later stage in mRNA metabolism. Here, we show that the presence of both Pab1p and Nab2p in reconstituted pre-mRNA 3′-end processing reactions has no stimulating nor inhibitory effect on poly(A) tail regulation. Importantly, the poly(A)-binding proteins are essential to protect the mature mRNA from being subjected to a second round of processing. We have determined which domains of Nab2p are important to control polyadenylation and found that the RGG-box work in conjunction with the two last essential CCCH-type zinc finger domains. Finally, we have tried to delineate the mechanism by which Nab2p performs its regulation function during polyadenylation: it likely forms a complex with poly(A) tails different from a simple linear deposit of proteins as it has been observed with Pab1p

Dual Requirement for Yeast hnRNP Nab2p in mRNA poly(A) Tail Length Control and Nuclear Export

Recent studies of mRNA export factors have provided additional evidence for a mechanistic link between mRNA 3′‐end formation and nuclear export. Here, we identify Nab2p as a nuclear poly(A)‐binding protein required for both poly(A) tail length control and nuclear export of mRNA. Loss of NAB2 expression leads to hyperadenylation and nuclear accumulation of poly(A)+ RNA but, in contrast to mRNA export mutants, these defects can be uncoupled in a nab2 mutant strain. Previous studies have implicated the cytoplasmic poly(A) tail‐binding protein Pab1p in poly(A) tail length control during polyadenylation. Although cells are viable in the absence of NAB2 expression when PAB1 is overexpressed, Pab1p fails to resolve the nab2Δ hyperadenylation defect even when Pab1p is tagged with a nuclear localization sequence and targeted to the nucleus. These results indicate that Nab2p is essential for poly(A) tail length control in vivo, and we demonstrate that Nab2p activates polyadenylation, while inhibiting hyperadenylation, in the absence of Pab1p in vitro. We propose that Nab2p provides an important link between the termination of mRNA polyadenylation and nuclear export

An essential role for Clp1 in assembly of polyadenylation complex CF IA and Pol II transcription termination

Polyadenylation is a co-transcriptional process that modifies mRNA 3′-ends in eukaryotes. In yeast, CF IA and CPF constitute the core 3′-end maturation complex. CF IA comprises Rna14p, Rna15p, Pcf11p and Clp1p. CF IA interacts with the C-terminal domain of RNA Pol II largest subunit via Pcf11p which links pre-mRNA 3′-end processing to transcription termination. Here, we analysed the role of Clp1p in 3′ processing. Clp1p binds ATP and interacts in CF IA with Pcf11p only. Depletion of Clp1p abolishes transcription termination. Moreover, we found that association of mutations in the ATP-binding domain and in the distant Pcf11p-binding region impair 3′-end processing. Strikingly, these mutations prevent not only Clp1p-Pcf11p interaction but also association of Pcf11p with Rna14p-Rna15p. ChIP experiments showed that Rna15p cross-linking to the 3′-end of a protein-coding gene is perturbed by these mutations whereas Pcf11p is only partially affected. Our study reveals an essential role of Clp1p in CF IA organization. We postulate that Clp1p transmits conformational changes to RNA Pol II through Pcf11p to couple transcription termination and 3′-end processing. These rearrangements likely rely on the correct orientation of ATP within Clp1p

Rôle de l'hnRNP Nab2p dans la régulation de la polyadénylation des ARNm chez la levure Saccharomyces cerevisiae

Dans le noyau des cellules eucaryotes, les pré-messagers transcits par l'ARN polymérase II subissent une maturation de leur extrémité 3'. Celle-ci participe à l'arrêt de leur propre synthèse et conditionne l'export des ARNm matures vers le cytoplasme où ils seront traduits en protéines. Cette modification post-transcriptionnelle se déroule en deux étapes : un clivage endonucléolytique site-spécifique et une polymérisation de résidus adénosine 5' monophosphate initiée à partir de l'extrémité 3' nouvellement générée. Chez l'organisme modèle Saccharomyces cerevisiae, la longueur de la queue poly (A) synthétisée lors de cette seconde étape est régulée et atteint une taille de 60 nucléotides mais les mécanismes mis en jeu sont mal définis. Les travaux rapportés dans cette thèse traitent essentiellement de la protéine Nab2p, membre fondateur d'une nouvelle famille de protéines eucaryotes à motif CCCH liant le poly (A). Par des approches de biochimie et de biologie moléculaire, j'ai pu mettre en évidence que Nab2p utilise conjointement ses domaines zinc finger et RGG-box pour réguler la polyadénylation lors de réactions de maturation en 3' des ARN reconstituées in vitro. Les résultats présentés suggèrent aussi que, de par le complexe qu'elle forme avec la queue poly (A) synthétisée, Nab2p empêche l'ARNm mature d'être de nouveau substrat des complexes de maturation en inhibant tout reclivage et en bloquant l'accès de l'extrémité 3' de l'ARN à la poly (A) polymérase canonique nucléaire. Cette thèse renforce des travaux antérieurs menés in vivo et indiquent que la protéine Nab2p est un acteur majeur du contrôle de la longueur des queues poly(A) des ARNm chez la levure.In the nucleus of eukaryotic cells, newly described mRNA precursors are processed at their 3' end. This biochemical process is functionally linked to transcription termination and is essential for proper mRNP assembly and export to the cytoplasm. 3' end formation occurs as a two-steps reaction : a site-specific endonucleolytic cleavage followed by polymerization of adenosine-5'-monophosphate residues initiated at the newly created 3'end. In the yeast Saccharomyces cerevisiae, the length of the poly (A) tail synthesized during this second step is controlled to reach 60 nucleotides on average, but the mechanisms involved are poorly detailed. The work reported in this thesis mostly focuses on Nab2p, founding member of a new class of CCCH-containing poly(A) binding proteins. Using biochemical and biomolecular approaches, I have discovered that the zinc finger domains of Nabb2p work in conjunction with its RGG-box region to regulate mRNA polyadenylation in reconstituted 3'-end processing reactions in vitro. Also, the results presented here suggest that Nab2p binding to the synthesized poly(A) tail prevents the mature mRNA from being re-processed by the 3'-end processing complexes. It inhibits re-cleavage and denies access of the poly(A) tail 3' end to the canonical nuclear poly(A) polymerase. This thesis reinforces previous studies conducted in vivo and highlights the major role played by Nab2p in mRNA poly(A) tail length control in yeast.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Maturation post-transcriptionnelle des ARN messagers chez la levure Saccharomyces cerevisiae (rôle du facteur de clivage et polyadénylation CF IA à travers l'étude de la sous-unité Clp1p)

Chez la levure S. cerevisiae, l'ajout d'une queue poly (A) en 3' des ARNm fait intervenir les facteurs CF IA, CPF et les protéines Nab4p et Nab2p. Le facteur CF IA est composé des protéines Rna1 4p, Rna1 5p, Pcf1 1p et C1p 1p. In vitro, des extraits protéiques obtenus à partir de souches mutantes c1p1 ont une activité de maturation en 3' déficiente : C1p 1p est requise lors des deux étapes de clivage et polyadénylation. Les facteurs CF IA présents dans différents contextes ont été purifiés. Les versions mutantes de C1p 1p et la protéine Pcf1 1p ne sont pas présentes dans ces fractions. Pourtant, des expériences de double-hybride révèlent que l'interaction entre la protéine Pcf1 1p et les formes mutantes de C1p 1p est altérée. Nos résultats suggèrent que C1p 1p serait impliquée dans la stabilité du facteur CF IA. Par ailleurs, nous montrons que C1p 1p et Pcf1 1p sont impliquées dans la maturation en 3' des snoRNAs, une autre classe de transcrits synthétisés par l'ARN polymérase II.In yeast, mRNAs acquire a poly(A) tail at their 3'ends. This involves the factors CF IA, CPF, and the proteins Nab4p and Nab2p. CF IA is a complex comprising Rna1 4p, Rna1 5p, Pcf1 1p and C1p 1p. The latter has been found to be essential for both the cleavage and polyadenylation steps. Mutations in the CLP1 gene indeed gave rise to extracts that were unable to perform in vitro pre-mRNA 3'-end processing. Purification of CF IA in mutant backgrounds for CLP1 showed that Pcf1p and the mutant Clp 1p protein no longer copurified with CF IA. Two-hybrid analyses additionnally showed that PCF11 and mutant c1p1 alleles failed to interact at different temperatures. Finally, we investigated C1p1p and Pcf1 1p putative role in snoRNA 3' end formation. We could show that repressed expression of either CLP1 or PCF11 impaired significantly termination of transcription of snoRNA independent units, strongly suggesting their implication in 3' end formation of this other class of RNA poll II transcripts.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

The Schizosaccharomyces Pombe Pla1 Gene Encodes a Poly(A) Polymerase and Can Functionally Replace Its Saccharomyces Cerevisiae Homologue

We have isolated the poly(A) polymerase (PAP) encoding gene pla1 [for poly(A) polymerase] from the fission yeast Schizosaccharomyces pombe. Protein sequence alignments with other poly(A) polymerases reveal that pla1 is more closely related to Saccharomyces cerevisiae PAP than to bovine PAP. The two yeast poly(A) polymerases share significant sequence homology not only in the generally conserved N-terminal part but also in the C-terminus. Furthermore, pla1 rescues a S.cerevisiae PAP1 disruption mutant. An extract from the complemented strain is active in the specific in vitro polyadenylation assay. In contrast, recombinant PLA1 protein can not replace bovine PAP in the mammalian in vitro polyadenylation assay. These results indicate a high degree of conservation of the polyadenylation machinery among the evolutionary diverged budding and fission yeast

The major yeast poly(A)-binding protein is associated with cleavage factor IA and functions in premessenger RNA 3′-end formation

Polyadenylation of premessenger RNAs occurs posttranscriptionally in the nucleus of eukaryotic cells by cleavage of the precursor and polymerization of adenosine residues. In the yeast Saccharomyces cerevisiae, the mature poly(A) tail ranges from 60 to 70 nucleotides. 3′-end processing can be reproduced in vitro with purified factors. The cleavage reaction requires cleavage factors I and II (CF I and CF II), whereas polyadenylation involves CF I, polyadenylation factor I (PFI), and poly(A) polymerase (Pap1p). CF I has recently been separated into two factors, CF IA and CF IB. We have independently purified CF IA and found that five polypeptides cofractionate with the activity. They include Rna14p, Rna15p, Pcf11p, a new protein called Clp1p, and remarkably, the major poly(A)-binding protein Pab1p. Extracts from strains where the PAB1 gene is mutated or deleted are active for cleavage but generate transcripts bearing abnormally long poly(A) tracts. Complementation with recombinant Pab1p not only restores the length of the poly(A) tails to normal, but also triggers a poly(A) shortening activity. In addition, a monoclonal Pab1p antibody prevents the formation of poly(A) tails in extracts or in a reconstituted system. Our data support the notion that Pab1p is involved in the length control of the poly(A) tails of yeast mRNAs and define a new essential function for Pab1p in the formation of mature mRNAs

Walter Keller (1938-2023): a tribute from his mentees

No abstrac

Pti1p and Ref2p found in association with the mRNA 3′ end formation complex direct snoRNA maturation

Eukaryotic RNA polymerase II transcribes precursors of mRNAs and of non-protein-coding RNAs such as snRNAs and snoRNAs. These RNAs have to be processed at their 3′ ends to be functional. mRNAs are matured by cleavage and polyadenylation that require a well-characterized protein complex. Small RNAs are also subject to 3′ end cleavage but are not polyadenylated. Here we show that two newly identified proteins, Pti1p and Ref2p, although they were found associated with the pre-mRNA 3′ end processing complex, are essential for yeast snoRNA 3′ end maturation. We also provide evidence that Pti1p probably acts by uncoupling cleavage and polyadenylation, and functions in coordination with the Nrd1p-dependent pathway for 3′ end formation of non-polyadenylated transcripts