Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 May;24(10):4196-206.
doi: 10.1128/MCB.24.10.4196-4206.2004.

Identification of factors regulating poly(A) tail synthesis and maturation

David A Mangus  1 , Mandy M Smith, Jennifer M McSweeney, Allan Jacobson
Affiliations

Identification of factors regulating poly(A) tail synthesis and maturation

David A Mangus et al. Mol Cell Biol. 2004 May.

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 fir1 Delta and pbp1 Delta 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).

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Mapping protein-protein interaction domains in Pbp1p and its interacting partners. Protein fragments used in two-hybrid analyses are denoted as bars, with numbers indicating the span of amino acids encoded by each construct. Proteins that interacted activated transcription of the HIS3 gene, producing resistance to the competitive inhibitor 3-AT. The results are expressed as the highest concentration of 3-AT (on plates of SC medium lacking His, Leu, and Trp) that still allowed substantial cellular growth; “no growth” indicates that cells could grow in the presence of histidine, but were unable to grow on medium lacking histidine (SC −Leu, −Trp, −His). (A) N-terminal and C-terminal truncations of Pbp1p were used to map the sites of interaction with factors identified in the two-hybrid screen. Each PBP1 fragment was tested against FIR1(281-925), UFD1-FL (full length), PBP4(93-185), DIG1(48-452), and empty GAL4(AD) vector. The PBP1(1-722) allele is identical to that previously designated PBP1-FL (32). (B) Fragments of interacting proteins identified in the two-hybrid screen with full-length Pbp1p.
FIG. 2.
FIG. 2.
The extent of in vitro polyadenylation is altered in extracts derived from fir1Δ, ref2Δ, and pbp1Δ strains. RNA processing extracts were prepared from the indicated strains and used for in vitro polyadenylation with a precleaved 183-nt CYC1 pre-mRNA. Radiolabeled ATP was used to monitor the reaction products. The reactions were terminated after 1 h, electrophoresed on 6% acrylamide gels, and visualized by autoradiography. M, radiolabeled DNA markers of indicated sizes (nucleotides).
FIG. 3.
FIG. 3.
Wild-type extracts reverse defects in poly(A) tail synthesis and maturation. RNA processing extracts prepared from pbp1Δ, fir1Δ, and ref2Δ strains were mixed with equal amounts of wild-type extracts for 30 min on ice and then used for in vitro polyadenylation of a precleaved CYC1 pre-mRNA. Reaction conditions and product analyses were identical to those described in the legend to Fig. 2.
FIG. 4.
FIG. 4.
Fir1p positively regulates poly(A) synthesis. (A) RNA processing extracts were prepared from wild-type and fir1Δ strains and used for in vitro polyadenylation of precleaved CYC1 pre-mRNA. Reactions, and their analysis, were identical to those depicted in Fig. 2, except that samples were taken at various times, as indicated. (B) The peak amount of polyadenylation from each reaction was determined by densitometry and graphed versus the time of incubation.
FIG. 5.
FIG. 5.
Ref2p negatively regulates poly(A) synthesis. (A) RNA processing extracts were prepared from wild-type and ref2Δ strains and used for in vitro polyadenylation of a precleaved CYC1 pre-mRNA as in Fig. 4. (B) The peak amount of polyadenylation was determined and plotted as in Fig. 4.
FIG. 6.
FIG. 6.
Extracts of fir1Δ/ref2Δ cells are phenotypically identical to ref2Δ extracts. (A) RNA processing extracts were prepared from wild-type and fir1Δ/ref2Δ strains and used for in vitro polyadenylation of a precleaved CYC1 pre-mRNA as in Fig. 4. (B) The peak amount of polyadenylation was determined and plotted as in Fig. 4.
FIG. 7.
FIG. 7.
Pbp1p is a negative regulator of poly(A) trimming. (A) RNA processing extracts were prepared from wild-type and pbp1Δ strains and used for in vitro polyadenylation of precleaved CYC1 pre-mRNA as in Fig. 4. (B) The peak amount of polyadenylation from each reaction was determined and plotted as in Fig. 4.
FIG. 8.
FIG. 8.
The absence of Pbp1p accelerates poly(A) shortening. (A) Fully adenylated, radiolabeled substrate was purified after incubating the precleaved CYC1 3′ RNA fragment in wild-type extract for 20 min and then incubated with wild-type and pbp1Δ extracts. Assay conditions were the same as those used for polyadenylation reactions except that labeled and unlabeled ATP were omitted. Samples were taken at various times, as indicated, and analyzed by gel electrophoresis and autoradiography. (B) The peak amount of poly(A) from each reaction was determined and plotted as in Fig. 4.
FIG. 9.
FIG. 9.
Pbp1p is a negative regulator of poly(A) nuclease. (A and C) RNA processing extracts were prepared from wild-type, pan2Δ, and pan2Δ/pbp1Δ strains and used for in vitro polyadenylation of precleaved CYC1 pre-mRNA as in Fig. 4. (B and D) The peak amount of polyadenylation from each reaction was determined and plotted as in Fig. 4.
FIG. 10.
FIG. 10.
Factors associated with poly(A) polymerase (Pap1p) and poly(A)-binding protein (Pab1p) act to regulate poly(A) tail synthesis and maturation. In this model, the protein interactions identified in this study are superimposed on the core factors (blue) known to be required for pre-mRNA 3′ processing. These new interactions are postulated to link CPF to the CFI subunits and to play a role in coordinating polyadenylation factors (yellow) with those involved in poly(A) trimming (red).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Amrani, N., M. E. Dufour, N. Bonneaud, and F. Lacroute. 1996. Mutations in STS1 suppress the defect in 3′ mRNA processing caused by the rna15-2 mutation in Saccharomyces cerevisiae. Mol. Gen. Genet. 252:552-562. - PubMed
    1. Amrani, N., M. Minet, M. Le Gouar, F. Lacroute, and F. Wyers. 1997. Yeast Pab1 interacts with Rna15 and participates in the control of the poly(A) tail length in vitro. Mol. Cell. Biol. 17:3694-3701. - PMC - PubMed
    1. Barabino, S. M., M. Ohnacker, and W. Keller. 2000. Distinct roles of two Yth1p domains in 3′-end cleavage and polyadenylation of yeast pre-mRNAs. EMBO J. 19:3778-3787. - PMC - PubMed
    1. Bartel, P., C. T. Chien, R. Sternglanz, and S. Fields. 1993. Elimination of false positives that arise in using the two-hybrid system. BioTechniques 14:920-924. - PubMed
    1. Bartel, P. L., and S. Fields. 1997. The yeast two-hybrid system. Oxford University Press, New York, N.Y.

Publication types

MeSH terms