A multisubunit factor, CstF, is required for polyadenylation of mammalian pre-mRNAs.

Takagaki, Yoshio; Manley, James L.; MacDonald, Clinton C.; Wilusz, Jeffrey; Shenk, Thomas

doi:10.1101/gad.4.12a.2112

Cited by 237 publications

(238 citation statements)

References 55 publications

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“…We reassembled the Rna14-Rna15 heterodimer (14), which echoes the mammalian CstF77-CstF64 pair (24). The direct interaction we observed between Pcf11 and Clp1 confirms a reported two-hybrid interaction (25) and suggests that the coimmunoprecipitation and copurification (4) of the human homologs of these proteins also may be caused by a direct interaction.…”

Section: Discussionsupporting

confidence: 57%

Five subunits are required for reconstitution of the cleavage and polyadenylation activities of Saccharomyces cerevisiae cleavage factor I

Größ

Moore

2001

Proc. Natl. Acad. Sci. U.S.A.

142

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Cleavage and polyadenylation of mRNA 3 ends in Saccharomyces cerevisiae requires several factors, one of which is cleavage factor I (CF I). Purification of CF I activity from yeast extract has implicated numerous proteins as functioning in both cleavage and͞or polyadenylation. Through reconstitution of active CF I from separately expressed and purified proteins, we show that CF I contains five subunits, Rna14, Rna15, Pcf11, Clp1, and Hrp1. These five are necessary and sufficient for reconstitution of cleavage activity in vitro when mixed with CF II, and for specific polyadenylation when mixed with polyadenylation factor I, purified poly(A) polymerase, and poly(A) binding protein. Analysis of the individual proteinprotein interactions supports an architectural model for CF I in which Pcf11 simultaneously interacts with Rna14, Rna15, and Clp1, whereas Rna14 bridges Rna15 and Hrp1.

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Section: Discussionsupporting

confidence: 57%

Five subunits are required for reconstitution of the cleavage and polyadenylation activities of Saccharomyces cerevisiae cleavage factor I

Größ

Moore

2001

Proc. Natl. Acad. Sci. U.S.A.

142

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“…While examining these differences, we discovered CstF-64 (8), which is a paralog of the 64,000 M r subunit of the cleavage stimulation factor (CstF-64) (9)(10)(11). CstF-64 is expressed in nuclei of early spermatogenic cells (5,12).…”

mentioning

confidence: 99%

Loss of polyadenylation protein τCstF-64 causes spermatogenic defects and male infertility

Dass

Tardif

Park

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

131

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Polyadenylation, the process of eukaryotic mRNA 3 end formation, is essential for gene expression and cell viability. Polyadenylation of male germ cell mRNAs is unusual, exhibiting increased alternative polyadenylation, decreased AAUAAA polyadenylation signal use, and reduced downstream sequence element dependence. CstF-64, the RNA-binding component of the cleavage stimulation factor (CstF), interacts with pre-mRNAs at sequences downstream of the cleavage site. In mammalian testes, meiotic XY-body formation causes suppression of X-linked CstF-64 expression during pachynema. Consequently, an autosomal paralog, CstF-64 (gene name Cstf2t), is expressed during meiosis and subsequent haploid differentiation. Here we show that targeted disruption of Cstf2t in mice causes aberrant spermatogenesis, specifically disrupting meiotic and postmeiotic development, resulting in male infertility resembling oligoasthenoteratozoospermia. Furthermore, the Cstf2t mutant phenotype displays variable expressivity such that spermatozoa show a broad range of defects. The overall phenotype is consistent with a requirement for CstF-64 in spermatogenesis as indicated by the significant changes in expression of thousands of genes in testes of Cstf2t ؊/؊ mice as measured by microarray. Our results indicate that, although the infertility in Cstf2t ؊/؊ males is due to low sperm count, multiple genes controlling many aspects of germ-cell development depend on CstF-64 for their normal expression. Finally, these transgenic mice provide a model for the study of polyadenylation in an isolated in vivo system and highlight the role of a growing family of testis-expressed autosomal retroposed variants of X-linked genes.spermatogenesis ͉ oligoasthenoteratozoospemia ͉ meiosis ͉ XY body ͉ meiotic sex chromosome inactivation P olyadenylation, the process of mRNA 3Ј end formation, is required for the synthesis, transport, translation, and stability of eukaryotic mRNAs (1, 2). Although polyadenylation is nearly universal, features of polyadenylation are different in mammalian male germ cells than in other tissues: male germ cell mRNAs exhibit increased alternative polyadenylation (3, 4), decreased use of the AAUAAA polyadenylation signal (5, 6), and reduced dependence on downstream sequence elements (DSEs) (7). These differences suggest a modified mechanism for polyadenylation in male germ cells.While examining these differences, we discovered CstF-64 (8), which is a paralog of the 64,000 M r subunit of the cleavage stimulation factor (CstF-64) (9-11). CstF-64 is expressed in nuclei of early spermatogenic cells (5, 12). However, because it is on the X chromosome, CstF-64 expression halts in pachytene spermatocytes because of meiotic sex chromosome inactivation (MSCI) (13). In contrast, CstF-64 expression begins in pachytene spermatocytes and continues in spermatocytes and early spermatids (refs. 5 and 12; summarized in Fig. 1). CstF-64 is the only known CstF-64 homolog expressed during male meiosis, thus making it a candidate to play a critical role in sper...

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“…CFI and CFII have not yet been well characterized but appear to be proteins of -110 and 135 kD, respectively, that are likely responsible for the cleavage of the pre-mRNA (Takagaki et al 1989). CstF consists of three subunits of 50, 64, and 77 kD (Takagaki et al 1990, 1992~ Gilmartin and Nevins 19911 Takagaki and Manley 1992 and interacts with CPSF to help specify the polyadenylation site. CPSF has three subunits, 160, 100, and 70 kD, and perhaps an additional subunit of -30 kD (Bienroth et al 1991~ Murthy and Manley 1992).…”

mentioning

confidence: 99%

Interaction between the U1 snRNP-A protein and the 160-kD subunit of cleavage-polyadenylation specificity factor increases polyadenylation efficiency in vitro.

Lutz¹,

Murthy²,

Schek³

et al. 1996

Genes Dev.

Self Cite

155

131

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We have previously shown that the U1 snRNP-A protein {U1A) interacts with elements in the SV40 late polyadenylation signal and that this association increases polyadenylation efficiency. It was postulated that this interaction occurs to facilitate protein-protein association between components of the U1 snRNP and proteins of the polyadenylation complex. We have now used GST fusion protein experiments, coimmunoprecipitations and Far Western blot analyses to demonstrate direct binding between U1A and the 160-kD subunit of cleavage--polyadenylation specificity factor (CPSF}. In addition, Western blot analyses of fractions from various stages of CPSF purification indicated that U1A copurified with CPSF to a point but could be separated in the highly purified fractions. These data suggest that UIA protein is not an integral component of CPSF but may be able to interact and affect its activity. In this regard, the addition of purified, recombinant U1A to polyadenylation reactions containing CPSF, polyIA) polymerase, and a precleaved RNA substrate resulted in concentration-dependent increases in both the level of polyadenylation and polylA} tail length. In agreement with the increase in polyadenylation efficiency caused by U1A, recombinant U1A stabilized the interaction of CPSF with the AAUAAA-containing substrate RNA in electrophoretic mobility shift experiments. These findings suggest that, in addition to its function in splicing, U1A plays a more global role in RNA processing through effects on polyadenylation.[ Moore et al. 1993; Sachs and Wahle 1993}. Splicing involves removal of intronic sequences and ligation of exons by a complex set of small nuclear ribonucleoprotein particles (snRNPsl and other factors known collectively as the spliceosome. Polyadenylation is the process by which the 3' end is formed through specific endonucleolytic cleavage of the precursor RNA and the addition of -250 adenosine residues. Both in vitro (Niwa et al. 1990;Berget 1991} and in vivo (Chiou et al. 1991;Nesic et al. 1993; Nesic and Maquat 1994} experiments 4These authors contributed equally to this work. SCorresponding author.have suggested that the processes of splicing and polyadenylation might be functionally linked. This proposal has been supported by our previous report that the U1 snRNP-A protein (U1A) interacts with elements in the SV40 late polyadenylation signal and that these interactions increase polyadenylation efficiency (Lutz and A1-wine 1994}. These data support the exon definition model of Berget and co-workers (for review, see Berget 1995}, which suggests that components of both the spliceosome and the polyadenylation complex may interact to define the last exon and affect the efficiencies of polyadenylation and last intron removal.There are currently five established mammalian factors comprising the complex that cleaves and polyadenylates substrate RNAs: cleavage-polyadenylation specificity factor (CPSF), cleavage stimulatory factor (CstF), polYIA) polymerase {PAP}, and cleavage factors I and II (CFI and CFII}...

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A multisubunit factor, CstF, is required for polyadenylation of mammalian pre-mRNAs.

Cited by 237 publications

References 55 publications

Five subunits are required for reconstitution of the cleavage and polyadenylation activities of Saccharomyces cerevisiae cleavage factor I

Five subunits are required for reconstitution of the cleavage and polyadenylation activities of Saccharomyces cerevisiae cleavage factor I

Loss of polyadenylation protein τCstF-64 causes spermatogenic defects and male infertility

Interaction between the U1 snRNP-A protein and the 160-kD subunit of cleavage-polyadenylation specificity factor increases polyadenylation efficiency in vitro.

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