Functional analysis of point mutations in the AAUAAA motif of the SV40 late polyadenylation signal

Wilusz, Jeffrey; Pettine, Stefan Mark; Shenk, Thomas

doi:10.1093/nar/17.10.3899

Cited by 80 publications

(44 citation statements)

References 47 publications

(67 reference statements)

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“…We also show that, in contrast to vertebrate systems, a point mutation in this sequence is partly recognized. It is noteworthy that in vertebrate in vivo (Montell et al 1983) and in vitro (Wilusz et al 1989) systems, a similar mutation would reduce cleavage by >95%. Apparently, the stringency for the AATAAA consensus sequence is much lower than in vertebrate systems.…”

Section: Discussionmentioning

confidence: 99%

“…Fifty-five percent of the transcripts were processed at the CaMV site and most of the remaining transcripts at the cryptic nos site (35%). It is noteworthy that in vertebrate in vivo (Montell et al 1983) and in vitro (Wilusz et al 1989) systems, a similar mutation would reduce cleavage by more than 95%.…”

Section: An Aataaa Sequence Is Required For Processing At the Camv Sitementioning

confidence: 99%

“…Point mutations in this sequence greatly reduce the efficiency of 3'-end formation in vivo (Fitzgerald and Shenk 1981;Montell et al 1983;Wickens and Stephenson 1984) and of both cleavage and polyadenylation in vitro Zarkower et al 1986;Conway and Wickens 1987;Wilusz et al 1989). In addition, a less well-conserved T-rich or TG-rich element, usually situated imiPresent address: Agriculture Canada Research Station, Vancouver, B.C., V6T 1X2 Canada.…”

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confidence: 99%

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A dissection of the cauliflower mosaic virus polyadenylation signal.

Sanfaçon

Brodmann²,

Hohn³

1991

Genes Dev.

101

107

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Section: Discussionmentioning

confidence: 99%

Section: An Aataaa Sequence Is Required For Processing At the Camv Sitementioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

A dissection of the cauliflower mosaic virus polyadenylation signal.

Sanfaçon

Brodmann²,

Hohn³

1991

Genes Dev.

101

107

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“…The cDNA contains 1584 bp up to the poly(A) tail. The composite cDNA contains a consensus polyadenylation signal (Wilusz et al, 1989) in the 3' untranslated region (UTR) 23 bp upstream to the poly(A) tail. The short 3' UTR was con®rmed by RT ± PCR amplification of the 3' end of cbl-3 from RNA from the CFPAC-1 and ZR75-1 cell lines utilizing an oligo dT primer (to both synthesize the ®rst strand cDNA and amplify the cDNA) and a sense primer within the coding region of the cDNA.…”

Section: Identi®cation and Cloning Of Cbl-3mentioning

confidence: 99%

cbl-3: a new mammalian cbl family protein

et al. 1999

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We have cloned a new human gene, cbl-3, which encodes a protein with marked homology to the cbl family of proteins. The predicted protein encoded by this gene retains the conserved phosphotyrosine binding domain (PTB) in the N-terminal and the zinc ®nger but is signi®cantly shorter (MW 52.5 kDa) than the other mammalian cbl proteins. The protein lacks the extensive proline rich domain and leucine zipper seen in c-cbl and cbl-b and structurally most resembles the C. elegans and Drosophila cbl proteins. The gene is ubiquitously expressed with highest expression in the aerodigestive tract, prostate, adrenal gland, and salivary gland. The protein is phosphorylated and recruited to the EGFR upon EGF stimulation and inhibits EGF stimulated MAP kinase activation. In comparison to the other mammalian cbl proteins (e.g. cbl-b), cbl-3 interacts with a restricted range of proteins containing Src Homology 3 regions. An alternatively spliced form of the cbl-3 protein was also identi®ed which deletes a critical region of the PTB domain and which does not interact with the EGFR nor inhibit EGF stimulated MAP kinase activation. These data demonstrate that cbl-3, a novel mammalian cbl protein, is a regulator of EGFR mediated signal transduction.

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“…This cleavage-polyadenylation event is mediated through two cis-acting sequence elements: a highly conserved hexanucleotide, AAUAAA, located 5 to 30 bases upstream of the cleavage site (8,12,22,27,35,37), and a less conserved U-or GU-rich stretch located downstream of the cleavage site (6,7,10,17,19,20,28,30,31). The selective recognition of alternative polyadenylation sites may play a role in the posttranscriptional regulation of gene expression (reviewed in reference 14).…”

mentioning

confidence: 99%

A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates.

Wilusz¹,

Shenk²,

Takagaki³

et al. 1990

Mol. Cell. Biol.

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A 64-kilodalton (kDa) polypeptide that is cross-linked by UV light specifically to polyadenylation substrate RNAs containing a functional AAUAAA element has been identified previously. Fractionated HeLa nuclear components that can be combined to regenerate efficient and accurate polyadenylation in vitro have now been screened for the presence of the 64-kDa protein. None of the individual components contained an activity which could generate the 64-kDa species upon UV cross-linking in the presence of substrate RNA. It was necessary to mix two components, cleavage stimulation factor and specificity factor, to reconstitute 64-kDa protein-RNA cross-linking. The addition of cleavage factors to this mixture very efficiently reconstituted the AAUAAAspecific 64-kDa protein-RNA interaction. The 64-kDa protein, therefore, is present in highly purified, reconstituted polyadenylation reactions. However, it is necessary to form a multicomponent complex to efficiently cross-link the protein to a substrate RNA.The maturation of the 3' ends of most RNA polymerase II transcripts involves an endonucleolytic cleavage event followed by the polymerization of 150 to 200 adenylate residues onto the newly formed terminus (reviewed in references 2 and 16). This cleavage-polyadenylation event is mediated through two cis-acting sequence elements: a highly conserved hexanucleotide, AAUAAA, located 5 to 30 bases upstream of the cleavage site (8,12,22,27,35,37), and a less conserved U-or GU-rich stretch located downstream of the cleavage site (6,7,10,17,19,20,28,30,31). The selective recognition of alternative polyadenylation sites may play a role in the posttranscriptional regulation of gene expression (reviewed in reference 14).Progress in understanding the mechanism of polyadenylation has been facilitated greatly by the development of accurate and faithful in vitro systems (15,24). The recognition of the polyadenylation signal occurs through the formation of a large, multicomponent complex. Native gel and density gradient analyses have shown that a 40 to 50S complex forms in an AAUAAA-dependent manner on substrate RNAs (13,25,32,33,39,40). Immunological and biochemical studies suggest that small nuclear RNAs play some role in this complex (1,11,13,(24)(25)(26). Label transfer experiments with UV light have identified proteins of 64 to 68 and 155 kilodaltons (kDa) which interact with RNAs that contain a polyadenylation signal (23, 38). Additionally, biochemical fractionation of the nuclear extract has identified several independent components which are required for reconstitution of in vitro processing (3, 4, 9, 18, 21, 34, 34a).We have now assayed partially purified polyadenylation components (34, 34a) for the presence of the 64-kDa protein by UV cross-linking. None of the individual fractions contained an activity that could be cross-linked to RNA to generate the 64-kDa species. However, a mixture of at least * Corresponding author. A flow chart of the biochemical purification steps performed is shown in Fig. 1. Details of the purificati...

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Functional analysis of point mutations in the AAUAAA motif of the SV40 late polyadenylation signal

Cited by 80 publications

References 47 publications

A dissection of the cauliflower mosaic virus polyadenylation signal.

A dissection of the cauliflower mosaic virus polyadenylation signal.

cbl-3: a new mammalian cbl family protein

A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates.

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