Separation and characterization of a poly(A) polymerase and a cleavage/specificity factor required for pre-mRNA polyadenylation

Takagaki, Yoshio; Ryner, Lisa; Manley, James L.

doi:10.1016/0092-8674(88)90411-4

Cited by 192 publications

(237 citation statements)

References 37 publications

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“…Lack of a specific ATP-dependent poly(A) addition reaction in the DE150 fraction (McDevitt et al, 1988;Gilmartin and Nevins, 1989), or in similar first-step purification fractions (Christofori and Keller, 1988;Takagaki et al 1988;Bardwell et al, 1990) has been reported previously. It is well established that the poly(A) addition reaction assayed with ATP requires a non-specific poly(A) polymerase (Christofori and Keller, 1989;Gilmartin and Nevins, 1989;Bardwell et al, 1990) and at least one, and possibly several, additional factors which recognize the AAUAAA sequence element (Christofori and Keller, 1988;Takagaki et al, 1988;Gilmartin and Nevins, 1989). To test if the ADP-dependent poly(A) addition reaction also requires multiple components, the crude nuclear extract was fractionated on a 15-35% glycerol gradient and the fractions were recombined and assayed for ADP-and ATP-supported polyadenylation.…”

Section: Resultsmentioning

confidence: 99%

“…2). The lack of ATP-supported poly(A) addition by the DE150 fraction (McDewitt et al, 1988;Gilmartin and Nevins, 1989) and by similar fractions from other purification schemes (Christofori and Keller, 1988;Takagaki et al, 1988) have been reported from several laboratories.…”

Section: Discussionmentioning

confidence: 99%

“…A second possible reason that ADP might act in the polyadenylation reaction concerns the reaction mechanism. It has been suggested (Takagaki et al, 1988) that the cleavage step in poly(A) addition requires ATP.…”

Section: Discussionmentioning

confidence: 99%

“…Loss of the ATP-supported reaction can be explained by removal of a factor, or factors, required for the specific poly(A) addition reaction (Christofori and Keller, 1988 ;Takagaki et al, 1988;Gilmartin and Nevins, 1989). These factors recognize the AAUAAA sequence element.…”

Section: Discussionmentioning

confidence: 99%

“…Polyadenylation of primary transcripts requires at least two steps : (a) a specific cleavage of the elongated transcript at a site downstream from the specific polyadenylation sequence element (AAUAAA) (Fitzgerald and Shenk, 1981 ; Gil and Proudfott, 1984;Hart et al, 1985;Zarkower et al, 1986); and (b) subsequent 3' poly(A) addition (Moore and Sharp, 1985;Zarkower et al, 1986). These reactions have been partially resolved and shown to require, in addition to a nonspecific poly(A) polymerase, a polymerase specificity factor (or factors) which recognizes the AAUAAA sequence element (Christofori and Keller, 1988;McDevitt et al, 1988;Takagaki et al, 1988;Gilmartin and Nevins, 1989), and one or more factors responsible for the cleavage process. The total number of factors required for the entire cleavage/poly(A) addition reaction is not known.…”

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confidence: 99%

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ADP is a substrate for the AAUAAA‐directed poly(A) addition reaction catalyzed by HeLa cell nuclear extracts

Lakota

Nelson

1991

European Journal of Biochemistry

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The specific poly(A) addition reaction catalyzed by crude nuclear extracts from HeLa cells can use ADP as efficiently as ATP as the donor of AMP residues. Both the ADP-and ATP-supported reactions require an intact upstream polyadenylation signal sequence element (AAUAAA). The mutated signal sequence (AACAAA) supports neither reaction. The ADP-supported poly(A) addition reaction can be resolved by glycerol gradient centrifugation of the crude nuclear extract into two components which are active when recombined but are inactive individually. The ATP-supported poly(A) addition is reconstituted by recombining the same gradient fractions, but the activity is lower than that supported by ADP, suggesting that an ATP-specific factor has been removed. A 150 mM KCl fraction DEAE-Sepharose of the nuclear extract, also devoid of the ATP-supported poly(A) addition reaction, retains a normal ADP-supported reaction. Together, these data show that ADP is a substrate for polyadenylation, and suggest that different factors might be required to induce ADP-or ATP-specificity in the poly(A) addition reaction.Most eucaryotic mRNAs are 3'-polyadenylated. Polyadenylation of primary transcripts requires at least two steps : (a) a specific cleavage of the elongated transcript at a site downstream from the specific polyadenylation sequence element (AAUAAA) (Fitzgerald and Shenk, 1981 ; Gil and Proudfott, 1984;Hart et al., 1985;Zarkower et al., 1986); and (b) subsequent 3' poly(A) addition (Moore and Sharp, 1985;Zarkower et al., 1986). These reactions have been partially resolved and shown to require, in addition to a nonspecific poly(A) polymerase, a polymerase specificity factor (or factors) which recognizes the AAUAAA sequence element (Christofori and Keller, 1988;McDevitt et al., 1988;Takagaki et al., 1988;Gilmartin and Nevins, 1989), and one or more factors responsible for the cleavage process. The total number of factors required for the entire cleavage/poly(A) addition reaction is not known.It is widely accepted that the specific (AAUAAA-requiring) poly(A) addition reaction utilizes ATP as the donor of AMP residues. However, the data showing ATP specificity for the reaction has been obtained only for the nonspecific poly(A) polymerase (Winters and Edmonds, 1973 a;Nevins and Joklik, 1977). Nevins and Joklik (1977) showed that adenylation of total HeLa cell RNA by a purified nonspecific polymerase is supported by ATP but not by GTP, CTP, UTP or ADP. Similarly, Winters and Edmonds (1973a) showed that adenylation of calf thymus RNA by a nonspecific calf Correspondence to J. Lakota, Department of Biochemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, SwedenAbbreviation. DE150 fraction, fraction obtained from a nuclear extract by elution from a DEAE-Sepharose column with 150 mM KCI.Enzyme. Poly(A) polymerase (EC 2.7.7.-).thymus poly(A) polymerase is supported specifically by ATP.In contrast, we demonstrate here that ADP is as good a substrate as ATP for the AAUAAA-directed poly(A) addition reaction. We also present ...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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ADP is a substrate for the AAUAAA‐directed poly(A) addition reaction catalyzed by HeLa cell nuclear extracts

Lakota

Nelson

1991

European Journal of Biochemistry

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In VitroRNASplicing in Mammalian Cell Extracts

Rooke

Underwood

2001

CP Cell Biology

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Almost every eukaryotic pre‐mRNA generated by RNA polymerase II transcription requires the removal of introns to create mRNA. The correct splicing of constitutive exons is thus critical for normal protein expression and function. Moreover, the removal of many introns by the spliceosome is controlled in a tissue‐specific or developmentally specific manner. In order to study RNA splicing at a biochemical level, it is necessary to employ an in vitro, or cell‐free, system. Cell‐free splicing systems require two main components: (1) an extract made from mammalian cell nuclei and (2) the introns and exons of the eukaryotic gene of interest. This minigene construct allows the synthesis of sufficient quantities of pre‐mRNA substrates in vitro, which are then incubated in the nuclear extract and analyzed for splicing. Nuclear extracts, first developed for studying transcription in vitro, are modified for splicing. This unit describes how to set up an in vitro splicing reaction using a mammalian nuclear extract derived from either cell line or tissue, and how to analyze the splicing reaction products.

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The end of the message: 3'– end processing leading to polyadenylated messenger RNA

Wahle

1992

BioEssays

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Almost all messenger RNAs carry a polyadenylate tail that is added in a post-transcriptional reaction. In the nuclei of animal cells, the 3'-end of the RNA is formed by endonucleolytic cleavage of the primary transcript at the site of poly(A) addition, followed by the polymerisation of the tail. The reaction depends on specific RNA sequences upstream as well as downstream of the polyadenylation site. Cleavage and polyadenylation can be uncoupled in vitro. Polyadenylation is carried out by poly(A) polymerase with the aid of a specificity factor that binds the polyadenylation signal AAUAAA. Several additional factors are required for the initial cleavage. A newly discovered poly(A)-binding protein stimulates poly(A) tail synthesis and may be involved in the control of tail length. Polyadenylation reactions different from this scheme, either in other organisms or under special physiological circumstances, are discussed.

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Separation and characterization of a poly(A) polymerase and a cleavage/specificity factor required for pre-mRNA polyadenylation

Cited by 192 publications

References 37 publications

ADP is a substrate for the AAUAAA‐directed poly(A) addition reaction catalyzed by HeLa cell nuclear extracts

ADP is a substrate for the AAUAAA‐directed poly(A) addition reaction catalyzed by HeLa cell nuclear extracts

In VitroRNASplicing in Mammalian Cell Extracts

The end of the message: 3'– end processing leading to polyadenylated messenger RNA

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