scholarly journals The Glc7 Phosphatase Subunit of the Cleavage and Polyadenylation Factor Is Essential for Transcription Termination on snoRNA Genes

2008 ◽  
Vol 29 (5) ◽  
pp. 577-587 ◽  
Author(s):  
Eduard Nedea ◽  
Demet Nalbant ◽  
Daniel Xia ◽  
Nathaniel T. Theoharis ◽  
Bernhard Suter ◽  
...  
Keyword(s):  
Transcription Termination ◽  
Cleavage And Polyadenylation ◽  
Polyadenylation Factor

scholarly journals The Essential N Terminus of the Pta1 Scaffold Protein Is Required for snoRNA Transcription Termination and Ssu72 Function but Is Dispensable for Pre-mRNA 3′-End Processing

2009 ◽  
Vol 29 (8) ◽  
pp. 2296-2307 ◽  
Author(s):  
Mohamed A. Ghazy ◽  
Xiaoyuan He ◽  
Badri Nath Singh ◽  
Michael Hampsey ◽  
Claire Moore
Keyword(s):  
Amino Acids ◽  
Rna Polymerase Ii ◽  
Transcription Termination ◽  
Inhibitory Effect ◽  
Mrna Cleavage ◽  
Cleavage And Polyadenylation ◽  
Essential Region ◽  
Polyadenylation Factor

ABSTRACT Saccharomyces cerevisiae Pta1 is a component of the cleavage/polyadenylation factor (CPF) 3′-end processing complex and functions in pre-mRNA cleavage, poly(A) addition, and transcription termination. In this study, we investigated the role of the N-terminal region of Pta1 in transcription and processing. We report that a deletion of the first 75 amino acids (pta1-Δ75) causes thermosensitive growth, while the deletion of an additional 25 amino acids is lethal. The pta1-Δ75 mutant is defective for snoRNA termination, RNA polymerase II C-terminal domain Ser5-P dephosphorylation, and gene looping but is fully functional for mRNA 3′-end processing. Furthermore, different regions of Pta1 interact with the CPF subunits Ssu72, Pti1, and Ysh1, supporting the idea that Pta1 acts as a scaffold to organize CPF. The first 300 amino acids of Pta1 are sufficient for interactions with Ssu72, which is needed for pre-mRNA cleavage. By the degron-mediated depletion of Pta1, we show that the removal of this essential region leads to a loss of Ssu72, yet surprisingly, in vitro cleavage and polyadenylation remain efficient. In addition, a fragment containing amino acids 1 to 300 suppresses 3′-end processing in wild-type extracts. These findings suggest that the amino terminus of Pta1 has an inhibitory effect and that this effect can be neutralized through the interaction with Ssu72.

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. 

Separation of factors required for cleavage and polyadenylation of yeast pre-mRNA

1992 ◽  
Vol 12 (8) ◽  
pp. 3470-3481
Author(s):  
J Chen ◽  
C Moore
Keyword(s):  
Cell Extract ◽  
Yeast Cells ◽  
Factor I ◽  
Initial Separation ◽  
Cleavage And Polyadenylation ◽  
Precursor Rna ◽  
A Site ◽  
Processing Factors ◽  
Polyadenylation Factor ◽  
Mammalian System

Cleavage and polyadenylation of yeast precursor RNA require at least four functionally distinct factors (cleavage factor I [CF I], CF II, polyadenylation factor I [PF I], and poly(A) polymerase [PAP]) obtained from yeast whole cell extract. Cleavage of precursor occurs upon combination of the CF I and CF II fractions. The cleavage reaction proceeds in the absence of PAP or PF I. The cleavage factors exhibit low but detectable activity without exogenous ATP but are stimulated when this cofactor is included in the reaction. Cleavage by CF I and CF II is dependent on the presence of a (UA)6 sequence upstream of the GAL7 poly(A) site. The factors will also efficiently cleave precursor with the CYC1 poly(A) site. This RNA does not contain a UA repeat, and processing at this site is thought to be directed by a UAG...UAUGUA-type motif. Specific polyadenylation of a precleaved GAL7 RNA requires CF I, PF I, and a crude fraction containing PAP activity. The PAP fraction can be replaced by recombinant PAP, indicating that this enzyme is the only factor in this fraction needed for the reconstituted reaction. The poly(A) addition step is also dependent on the UA repeat. Since CF I is the only factor necessary for both cleavage and poly(A) addition, it is likely that this fraction contains a component which recognizes processing signals located upstream of the poly(A) site. The initial separation of processing factors in yeast cells suggests both interesting differences from and similarities to the mammalian system.

Poly(A) polymerase purified from HeLa cell nuclear extract is required for both cleavage and polyadenylation of pre-mRNA in vitro

1989 ◽  
Vol 9 (1) ◽  
pp. 193-203
Author(s):  
G Christofori ◽  
W Keller
Keyword(s):  
Hela Cell ◽  
Nuclear Extract ◽  
Cleavage Activity ◽  
Hela Cell Nuclear Extract ◽  
Cleavage And Polyadenylation ◽  
A Site ◽  
Polyadenylation Factor ◽  
Polyadenylated Mrna ◽  
Cell Nuclear Extract

We have partially purified a poly(A) polymerase (PAP) from HeLa cell nuclear extract which is involved in the 3'-end formation of polyadenylated mRNA. PAP had a molecular weight of approximately 50 to 60 kilodaltons. In the presence of manganese ions, PAP was able to polyadenylate RNA nonspecifically. However, in the presence of magnesium ions PAP required the addition of a cleavage and polyadenylation factor to specifically polyadenylate pre-mRNAs that contain an intact AAUAAA sequence and end at the poly(A) addition site (precleaved RNA substrates). The purified fraction containing PAP was also required in combination with a cleavage and polyadenylation factor and a cleavage factor for the correct cleavage at the poly(A) site of pre-mRNAs. Since the two activities of the PAP fractions, PAP and cleavage activity, could not be separated by extensive purification, we concluded that the two activities are contained in a single component, a PAP that is also required for the specific cleavage preceding the polyadenylation of pre-mRNA.

scholarly journals Identification of Factors Regulating Poly(A) Tail Synthesis and Maturation

2004 ◽  
Vol 24 (10) ◽  
pp. 4196-4206 ◽  
Author(s):  
David A. Mangus ◽  
Mandy M. Smith ◽  
Jennifer M. McSweeney ◽  
Allan Jacobson
Keyword(s):  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Genes Encoding ◽  
Mitogen Activated Protein ◽  
Regulatory Complex ◽  
Cleavage And Polyadenylation ◽  
Mrna Polyadenylation ◽  
Two Hybrid ◽  
Polyadenylation Factor

ABSTRACT Posttranscriptional maturation of the 3′ end of eukaryotic pre-mRNAs occurs as a three-step pathway involving site-specific cleavage, polymerization of a poly(A) tail, and trimming of the newly synthesized tail to its mature length. While most of the factors essential for catalyzing these reactions have been identified, those that regulate them remain to be characterized. Previously, we demonstrated that the yeast protein Pbp1p associates with poly(A)-binding protein (Pab1p) and controls the extent of mRNA polyadenylation. To further elucidate the function of Pbp1p, we conducted a two-hybrid screen to identify factors with which it interacts. Five genes encoding putative Pbp1p-interacting proteins were identified, including (i) FIR1/PIP1 and UFD1/PIP3, genes encoding factors previously implicated in mRNA 3′-end processing; (ii) PBP1 itself, confirming directed two-hybrid results and suggesting that Pbp1p can multimerize; (iii) DIG1, encoding a mitogen-activated protein kinase-associated protein; and (iv) PBP4 (YDL053C), a previously uncharacterized gene. In vitro polyadenylation reactions utilizing extracts derived from fir1Δ and pbp1Δ cells and from cells lacking the Fir1p interactor, Ref2p, demonstrated that Pbp1p, Fir1p, and Ref2p are all required for the formation of a normal-length poly(A) tail on precleaved CYC1 pre-mRNA. Kinetic analyses of the respective polyadenylation reactions indicated that Pbp1p is a negative regulator of poly(A) nuclease (PAN) activity and that Fir1p and Ref2p are, respectively, a positive regulator and a negative regulator of poly(A) synthesis. We suggest a model in which these three factors and Ufd1p are part of a regulatory complex that exploits Pab1p to link cleavage and polyadenylation factors of CFIA and CFIB (cleavage factors IA and IB) to the polyadenylation factors of CPF (cleavage and polyadenylation factor).

scholarly journals Codon usage biases co-evolve with transcription termination machinery to suppress premature cleavage and polyadenylation

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Zhipeng Zhou ◽  
Yunkun Dang ◽  
Mian Zhou ◽  
Haiyan Yuan ◽  
Yi Liu
Keyword(s):  
Protein Expression ◽  
Codon Usage ◽  
Codon Usage Bias ◽  
Transcription Termination ◽  
Model Organism ◽  
Open Reading Frames ◽  
Strong Negative Correlation ◽  
Rare Codons ◽  
Cleavage And Polyadenylation ◽  
Mouse Cells

Codon usage biases are found in all genomes and influence protein expression levels. The codon usage effect on protein expression was thought to be mainly due to its impact on translation. Here, we show that transcription termination is an important driving force for codon usage bias in eukaryotes. Using Neurospora crassa as a model organism, we demonstrated that introduction of rare codons results in premature transcription termination (PTT) within open reading frames and abolishment of full-length mRNA. PTT is a wide-spread phenomenon in Neurospora, and there is a strong negative correlation between codon usage bias and PTT events. Rare codons lead to the formation of putative poly(A) signals and PTT. A similar role for codon usage bias was also observed in mouse cells. Together, these results suggest that codon usage biases co-evolve with the transcription termination machinery to suppress premature termination of transcription and thus allow for optimal gene expression.

scholarly journals Direct Interactions between the Paf1 Complex and a Cleavage and Polyadenylation Factor Are Revealed by Dissociation of Paf1 from RNA Polymerase II

2008 ◽  
Vol 7 (7) ◽  
pp. 1158-1167 ◽  
Author(s):  
Kristen Nordick ◽  
Matthew G. Hoffman ◽  
Joan L. Betz ◽  
Judith A. Jaehning
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Direct Interaction ◽  
Pol Ii ◽  
Phosphorylated Form ◽  
Cleavage And Polyadenylation ◽  
Paf1 Complex ◽  
Processing Factors ◽  
Polyadenylation Factor ◽  
Transcription Cycle

ABSTRACT The Paf1 complex (Paf1, Ctr9, Cdc73, Rtf1, and Leo1) is normally associated with RNA polymerase II (Pol II) throughout the transcription cycle. However, the loss of either Rtf1 or Cdc73 results in the detachment of the Paf1 complex from Pol II and the chromatin form of actively transcribed genes. Using functionally tagged forms of the Paf1 complex factors, we have determined that, except for the more loosely associated Rtf1, the remaining components stay stably associated with one another in an RNase-resistant complex after dissociation from Pol II and chromatin. The loss of Paf1, Ctr9, or to a lesser extent Cdc73 or Rtf1 results in reduced levels of serine 2 phosphorylation of the Pol II C-terminal domain and in increased read through of the MAK21 polyadenylation site. We found that the cleavage and polyadenylation factor Cft1 requires the Pol II-associated form of the Paf1 complex for full levels of interaction with the serine 5-phosphorylated form of Pol II. When the Paf1 complex is dissociated from Pol II, a direct interaction between Cft1 and the Paf1 complex can be detected. These results are consistent with the Paf1 complex providing a point of contact for recruitment of 3′-end processing factors at an early point in the transcription cycle. The lack of this connection helps to explain the defects in 3′-end formation observed in the absence of Paf1.

scholarly journals Functional Analysis of Yeast snoRNA and snRNA 3′-End Formation Mediated by Uncoupling of Cleavage and Polyadenylation

2002 ◽  
Vol 22 (5) ◽  
pp. 1379-1389 ◽  
Author(s):  
Mariangela Morlando ◽  
Paolo Greco ◽  
Bernhard Dichtl ◽  
Alessandro Fatica ◽  
Walter Keller ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Sequence Analysis ◽  
Genetic Analysis ◽  
Small Rnas ◽  
Essential Role ◽  
Factor Activity ◽  
Noncoding Regions ◽  
Cleavage And Polyadenylation ◽  
Polyadenylation Factor

ABSTRACT Many nuclear and nucleolar small RNAs are accumulated as nonpolyadenylated species and require 3′-end processing for maturation. Here, we show that several genes coding for box C/D and H/ACA snoRNAs and for the U5 and U2 snRNAs contain sequences in their 3′ portions which direct cleavage of primary transcripts without being polyadenylated. Genetic analysis of yeasts with mutations in different components of the pre-mRNA cleavage and polyadenylation machinery suggests that this mechanism of 3"-end formation requires cleavage factor IA (CF IA) but not cleavage and polyadenylation factor activity. However, in vitro results indicate that other factors participate in the reaction besides CF IA. Sequence analysis of snoRNA genes indicated that they contain conserved motifs in their 3" noncoding regions, and mutational studies demonstrated their essential role in 3"-end formation. We propose a model in which CF IA functions in cleavage and polyadenylation of pre-mRNAs and, in combination with a different set of factors, in 3"-end formation of nonpolyadenylated polymerase II transcripts.

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