The Yeast Pan2 Protein Is Required for Poly(A)-binding Protein-stimulated Poly(A)-nuclease Activity

Boeck, Ronald; Tarun, Salvador Jr.; Rieger, Michael A.; Deardorff, Julie A.; Müller-Auer, S.; Sachs, Alan B.

doi:10.1074/jbc.271.1.432

Cited by 164 publications

(128 citation statements)

References 17 publications

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“…Using the previously published consensus for human PABC (Table III), we searched the S. cerevisiae genome for related sequences that might bind to yPABC. Among proteins known to interact with Pab1p, we identified residues 234 -250 from Pan1p (29,30), two regions from Pbp1p (residues 308 -327 and 376 -399) (11), and a peptide derived from the N terminus of RF3 (residues 106 -122) ( Table II). None of these peptides bound to yPABC, as determined by the absence of chemical shift changes in 1 H, 15 N correlation spectra.…”

Section: Resultsmentioning

confidence: 99%

Solution Structure of the Orphan PABC Domain fromSaccharomyces cerevisiae Poly(A)-binding Protein

Kozlov¹,

Siddiqui²,

Coillet-Matillon³

et al. 2002

Journal of Biological Chemistry

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We have determined the solution structure of the PABC domain from Saccharomyces cerevisiae Pab1p and mapped its peptide-binding site. PABC domains are peptide binding domains found in poly(A)-binding proteins (PABP) and are a subset of HECT-family E3 ubiquitin ligases (also known as hyperplastic discs proteins (HYDs)). In mammals, the PABC domain of PABP functions to recruit several different translation factors to the mRNA poly(A) tail. PABC domains are highly conserved, with high specificity for peptide sequences of roughly 12 residues with conserved alanine, phenylalanine, and proline residues at positions 7, 10, and 12. Compared with human PABP, the yeast PABC domain is missing the first ␣ helix, contains two extra amino acids between helices 2 and 3, and has a strongly bent Cterminal helix. These give rise to unique peptide binding specificity wherein yeast PABC binds peptides from Paip2 and RF3 but not Paip1. Mapping of the peptidebinding site reveals that the bend in the C-terminal helix disrupts binding interactions with the N terminus of peptide ligands and leads to greatly reduced binding affinity for the peptides tested. No high affinity or natural binding partners from S. cerevisiae could be identified by sequence analysis of known PABC ligands. Comparison of the three known PABC structures shows that the features responsible for peptide binding are highly conserved and responsible for the distinct but overlapping binding specificities.

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Section: Resultsmentioning

confidence: 99%

Solution Structure of the Orphan PABC Domain fromSaccharomyces cerevisiae Poly(A)-binding Protein

Kozlov¹,

Siddiqui²,

Coillet-Matillon³

et al. 2002

Journal of Biological Chemistry

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“…Pab1p alleles that fail to interact with Pan3p have long poly(A) tails. As noted above, pab1 mutations cause a significant increase in the average steady-state poly(A) tail length of total cellular mRNA (32), a defect thought to be at least partially attributable to the inability of the mutant Pab1p proteins to stimulate PAN activity (7,9,21). To determine if the loss of Pan3p binding to Pab1p resulted in similar defects in poly(A) tail maturation, Pan3p-noninteracting pab1 alleles were introduced into full-length PAB1 constructs and poly(A) tail lengths associated with total cellular mRNA were analyzed.…”

Section: Identification Of Pan3p Interaction Mutations Within the Pabmentioning

confidence: 99%

Positive and Negative Regulation of Poly(A) Nuclease

Mangus

Evans

Agrin

et al. 2004

Molecular and Cellular Biology

102

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With rare exceptions, mRNAs whose synthesis originates within nuclei contain a 3Ј poly(A) tail. Poly(A) tracts are not encoded within genes but are added to nascent pre-mRNAs in a processing reaction that involves site-specific cleavage and subsequent polyadenylation (11,16,40,42). In Saccharomyces cerevisiae, newly synthesized poly(A) tails of different transcripts are relatively homogeneous, with their final lengths determined by the combined actions of poly(A) polymerase holoenzyme (Pap1p and Fip1p), poly(A)-binding protein (Pab1p), poly(A) nuclease (PAN), and the Pab1p-associated factor, Pbp1p (10,24,43).Poly(A) tracts are generally bound by Pab1p, a highly conserved protein with four RNA recognition motifs (RRMs) connected to a carboxy-terminal helical domain via a prolineand methionine-rich segment (25,31). Association of Pab1p with poly(A) requires a minimal binding site of 12 adenosines, and multiple molecules can bind via RRMs 1 and 2 to the same poly(A) tract, spaced approximately 25 nucleotides apart (1, 2, 31, 33). In yeast, the relatively abundant 70-kDa poly(A)-binding protein is encoded by the PAB1 gene. Mutations in PAB1 cause a significant increase in the average steady-state poly(A) tail length of total cellular mRNA (32), and these effects have been attributed to two apparently nuclear functions of Pab1p: the regulation of a switch between processive and distributive activities in poly(A) polymerase (43) and the stimulation of PAN activity (7, 9, 21).Yeast poly(A) tails are initially synthesized to default lengths of 70 to 90 A's and then trimmed to mRNA-specific lengths by PAN. Analyses of three different mRNAs indicate that such trimmed tails have lengths ranging from 55 to 71 A's (8). PAN, a Pab1p-dependent 3Ј to 5Ј poly(A) exoribonuclease, requires magnesium, releases AMP as a product, and is regulated by cis-acting mRNA sequences (21). Purified PAN contains two proteins which are essential for nuclease activity: Pan2p is a 127-kDa protein with homology to the RNase T family of 3Ј35Ј exoribonucleases, while Pan3p is a 76-kDa protein which apparently acts as a positive activator of PAN activity (8). Deletion of PAN2 and/or PAN3 eliminates poly(A) nuclease activity but does not hinder cell growth. Physical interaction between Pan2p and Pan3p has been inferred from coimmunoprecipitation and two-hybrid analyses of the full-length proteins (9).Pbp1p (Pab1p-binding protein 1) specifically interacts with a 74-amino-acid segment encompassing the proline-and methionine-rich domain of the Pab1p C terminus. PBP1 is not essential for viability, but its disruption can suppress the lethality associated with a PAB1 deletion (23). In the absence of Pbp1p, 3Ј termini of pre-mRNAs are properly cleaved but receive poly(A) tails that are, on average, 15 to 30 nucleotides shorter than normal (23,25). In vitro polyadenylation reactions using extracts from wild-type and pbp1⌬ strains demonstrated that the mutant extracts initially produced full-length tails equivalent to their wild-type counterparts but subseque...

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“…A poly(A)-specific ribonuclease has been purified from calf thymus (8) and has very similar enzymatic properties to the activity in HeLa cells (9). In yeast there are two deadenylase complexes, Pan2p/Pan3p (41,42) and Ccr4p/Pop2p/Notp (43). Ccr4p is now thought to be the major deadenylase in yeast (43), and a human homologue has been cloned (10).…”

Section: P38 Mapk Inhibits Are-directed Deadenylationmentioning

confidence: 99%

p38 Mitogen-activated Protein Kinase Stabilizes mRNAs That Contain Cyclooxygenase-2 and Tumor Necrosis Factor AU-rich Elements by Inhibiting Deadenylation

Dean

Sarsfield

Tsounakou

et al. 2003

Journal of Biological Chemistry

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AU-rich elements (AREs) in 3 -untranslated regions of mRNAs confer instability. They target mRNAs for rapid deadenylation and degradation and may enhance decapping. The p38 MAPK pathway stabilizes many otherwise unstable ARE-containing mRNAs encoding proteins involved in inflammation; however, the mRNA decay step(s) regulated by the signaling pathway are unknown. To investigate whether it regulates deadenylation or the decay of the mRNA body, we used a tetracycline-regulated ␤-globin mRNA reporter system to transcribe pulses of mRNA of uniform length. We measured on Northern gels the migration of reporter mRNAs isolated from cells transfected only with reporter plasmid or co-transfected with an active mutant of MAPK kinase-6, and treated either with or without the p38 MAPK inhibitor SB 203580. Differences in migration were shown by RNase H mapping with oligo(dT) to be due to poly(A) shortening. Insertion of an ARE into the ␤-globin reporter mRNA promoted rapid deadenylation and decay of hypo-adenylated reporter mRNA. p38 MAPK activation inhibited the deadenylation of reporter mRNAs containing either the cyclooxygenase-2 or tumor necrosis factor AREs. The regulation of deadenylation by p38 MAPK was found to be specific because deadenylation of the ␤-globin reporter mRNA either lacking an ARE or containing the c-Myc 3 -untranslated region (which is not p38 MAPK-responsive) was unaffected by p38 MAPK. It was concluded that the p38 MAPK pathway predominantly regulates deadenylation, rather than decay of the mRNA body, and this provides an explanation for why p38 MAPK regulates mRNA stability in some situations and translation in others.

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The Yeast Pan2 Protein Is Required for Poly(A)-binding Protein-stimulated Poly(A)-nuclease Activity

Cited by 164 publications

References 17 publications

Solution Structure of the Orphan PABC Domain fromSaccharomyces cerevisiae Poly(A)-binding Protein

Solution Structure of the Orphan PABC Domain fromSaccharomyces cerevisiae Poly(A)-binding Protein

Positive and Negative Regulation of Poly(A) Nuclease

p38 Mitogen-activated Protein Kinase Stabilizes mRNAs That Contain Cyclooxygenase-2 and Tumor Necrosis Factor AU-rich Elements by Inhibiting Deadenylation

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