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. 

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.

Transcription termination at the chicken beta H-globin gene

1988 ◽  
Vol 8 (12) ◽  
pp. 5369-5377
Author(s):  
T M Pribyl ◽  
H G Martinson
Keyword(s):  
Rna Polymerase Ii ◽  
Inverted Repeat ◽  
Transcription Termination ◽  
Globin Gene ◽  
H Gene ◽  
Delta G ◽  
A Site ◽  
Cat Expression

We characterized the transcription termination region of the chicken beta H-globin gene. First we located the region by nuclear runon transcription in vitro. Then we sequenced and subcloned it into a chloramphenicol acetyltransferase (CAT) expression vector for assay in vivo. The region of beta H termination contains two interesting elements located about 1 kilobase downstream of the beta H gene poly(A) site. Either element alone can block CAT expression if inserted between the promoter and the poly(A) site of the cat gene in pRSVcat. The first element in the termination region is an unusually large inverted repeat in the DNA (delta G = -71 kcal). The second element, 200 base pairs further downstream, is an RNA polymerase II promoter which directs transcription back upstream on the complementary strand. This transcription converges on and collides with that from the beta H gene at or near the inverted repeat where transcription from both directions stops. We propose that the inverted repeat is a strong pause site which positions the converging polymerases for mutual site-specific termination.

scholarly journals Studies of the 5′ Exonuclease and Endonuclease Activities of CPSF-73 in Histone Pre-mRNA Processing

2008 ◽  
Vol 29 (1) ◽  
pp. 31-42 ◽  
Author(s):  
Xiao-cui Yang ◽  
Kelly D. Sullivan ◽  
William F. Marzluff ◽  
Zbigniew Dominski
Keyword(s):  
Cleavage Site ◽  
Complete Removal ◽  
Cleavage Product ◽  
Endonucleolytic Cleavage ◽  
Subsequent Degradation ◽  
U7 Snrnp ◽  
Space Requirements ◽  
Polyadenylation Factor

ABSTRACT Processing of histone pre-mRNA requires a single 3′ endonucleolytic cleavage guided by the U7 snRNP that binds downstream of the cleavage site. Following cleavage, the downstream cleavage product (DCP) is rapidly degraded in vitro by a nuclease that also depends on the U7 snRNP. Our previous studies demonstrated that the endonucleolytic cleavage is catalyzed by the cleavage/polyadenylation factor CPSF-73. Here, by using RNA substrates with different nucleotide modifications, we characterize the activity that degrades the DCP. We show that the degradation is blocked by a 2′-O-methyl nucleotide and occurs in the 5′-to-3′ direction. The U7-dependent 5′ exonuclease activity is processive and continues degrading the DCP substrate even after complete removal of the U7-binding site. Thus, U7 snRNP is required only to initiate the degradation. UV cross-linking studies demonstrate that the DCP and its 5′-truncated version specifically interact with CPSF-73, strongly suggesting that in vitro, the same protein is responsible for the endonucleolytic cleavage of histone pre-mRNA and the subsequent degradation of the DCP. By using various RNA substrates, we define important space requirements upstream and downstream of the cleavage site that dictate whether CPSF-73 functions as an endonuclease or a 5′ exonuclease. RNA interference experiments with HeLa cells indicate that degradation of the DCP does not depend on the Xrn2 5′ exonuclease, suggesting that CPSF-73 degrades the DCP both in vitro and in vivo.

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 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 The nucleosome DNA entry-exit site is important for transcription termination and prevention of pervasive transcription

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
A Elizabeth Hildreth ◽  
Mitchell A Ellison ◽  
Alex M Francette ◽  
Julia M Seraly ◽  
Lauren M Lotka ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Nucleosome Occupancy ◽  
Transcription Termination ◽  
Antisense Transcription ◽  
Exit Site ◽  
Genomic Changes ◽  
Sense Strand ◽  
Native Sequence

Compared to other stages in the RNA polymerase II transcription cycle, the role of chromatin in transcription termination is poorly understood. We performed a genetic screen in Saccharomyces cerevisiae to identify histone mutants that exhibit transcriptional readthrough of terminators. Amino acid substitutions identified by the screen map to the nucleosome DNA entry-exit site. The strongest H3 mutants revealed widespread genomic changes, including increased sense-strand transcription upstream and downstream of genes, increased antisense transcription overlapping gene bodies, and reduced nucleosome occupancy particularly at the 3’ ends of genes. Replacement of the native sequence downstream of a gene with a sequence that increases nucleosome occupancy in vivo reduced readthrough transcription and suppressed the effect of a DNA entry-exit site substitution. Our results suggest that nucleosomes can facilitate termination by serving as a barrier to transcription and highlight the importance of the DNA entry-exit site in broadly maintaining the integrity of the transcriptome.

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 Cell-cycle-specific transcription termination within the human histone H3.3 gene is correlated with specific protein–DNA interactions

1997 ◽  
Vol 69 (2) ◽  
pp. 101-110 ◽  
Author(s):  
ALAN TAYLOR ◽  
LIQUN ZHANG ◽  
JOHN HERRMANN ◽  
BEI WU ◽  
LARRY KEDES ◽  
...  
Keyword(s):  
Protein Binding ◽  
Rna Polymerase Ii ◽  
Transcription Termination ◽  
Transcription Elongation ◽  
Specific Protein ◽  
Binding Activity ◽  
Mobility Shift ◽  
Nuclear Extracts

In vitro studies using highly purified calf thymus RNA polymerase II and a fragment spanning the first intron of H3.3 as template DNA have demonstrated the existence of a strong transcription termination site consisting of thymidine stretches. In this study, nuclear run-on experiments have been performed to assess the extent to which transcription elongation is blocked in vivo using DNA probes corresponding to regions 5′ and 3′ of the in vitro termination sites. These studies suggest that H3.3 expression is stimulated following the inhibition of DNA synthesis through the elimination of the transcription elongation block. Interestingly, both the in vivo and in vitro experiments have revealed that the transcriptional block/termination sites are positioned immediately downstream of a 73 bp region that has been over 90% conserved between the chicken and human H3.3 genes. The extreme conservation of this intronic region suggests a possible role in maintaining cis-acting function. Electrophoretic mobility shift experiments show that HeLa cell nuclear extracts contain protein factors that bind specifically to the region of transcription elongation block. Furthermore, we demonstrate a correlation between the protein binding activity and the transcriptional block in cells that have been either arrested at the initiation of S phase or were replication-interrupted by hydroxyurea. DNA footprinting experiments indicate that the region of protein binding is at the 3′ end of the conserved region and overlaps with one of the three in-vitro-mapped termination sites.

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.

Visualizing transcription in real-time

2008 ◽  
Vol 3 (1) ◽  
pp. 11-18 ◽  
Author(s):  
Yehuda Brody ◽  
Yaron Shav-Tal
Keyword(s):  
Rna Polymerase Ii ◽  
Living Cells ◽  
Messenger Rnas ◽  
Protein Coding ◽  
Upstream Gene ◽  
Structural Modifications ◽  
Protein Coding Genes ◽  
Transcriptional Process ◽  
Kinetics Of

AbstractThe transcriptional process is at the center of the gene expression pathway. In eukaryotes, the transcription of protein-coding genes into messenger RNAs is performed by RNA polymerase II. This enzyme is directed to bind at upstream gene sequences by the aid of transcription factors that assemble transcription-competent complexes. A series of biochemical and structural modifications render the polymerase transcriptionally active so that it can proceed from an initiation state into a functional elongating phase. Recent experimental efforts have attempted to visualize these processes as they take place on genes in living cells and to quantify the kinetics of in vivo transcription.

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