The human U1 snRNP-Specific U1A protein inhibits polyadenylation of its own pre-mRNA

Boelens, Wilbert C.; Jansen, Erik; Venrooij, Walther J. van; Stripecke, Renata; Mattaj, Iain W.; Gunderson, Samuel

doi:10.1016/0092-8674(93)90577-d

Cited by 202 publications

(212 citation statements)

References 60 publications

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“…The occurrence of this inhibition may reflect an inhibitory effect of free U1A protein on PAP similar to that suggested for the autoregulation of polyadenylation of U1A's own mRNA (Boelens et al 1993;van Gelder et al 1993;Gunderson et al 1994). This involves two molecules of free U1A protein binding through their RRM1 domains to two adjacent sites upstream of the AAUAAA on the pre-mRNA encoding U1A protein (van Gelder et al 1993;Gunderson et al 1994).…”

Section: Genes and Developmentmentioning

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.

154

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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Section: Genes and Developmentmentioning

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.

154

131

View full text Add to dashboard Cite

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“…Recombinant Proteins-Untagged recombinant wild type U1A and U1A were purified from Escherichia coli as described previously (30). Recombinant bovine poly(A) polymerase, tagged at the C terminus with 6 histidines, was purified from E. coli on Ni 2ϩ -NTA.…”

Section: Methodsmentioning

confidence: 99%

Sequences Adjacent to the 5′ Splice Site Control U1A Binding Upstream of the IgM Heavy Chain Secretory Poly(A) Site

Phillips

Gunderson

2003

Journal of Biological Chemistry

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We have recently shown that the stability of the alternatively expressed immunoglobulin M heavy chain secretory mRNA is developmentally regulated by U1A. U1A binds novel non-consensus sites upstream of the secretory poly(A) site and inhibits poly(A) tail addition in undifferentiated cells. U1A's dependence for binding and function upon a stem-loop structure has been extensively characterized for the consensus sites. We therefore probed the structure surrounding the novel U1A binding sites. We show that two of the three novel binding sites represent the major single-stranded regions upstream of the secretory poly(A) site, consistent with a major role at this site. The strength of binding and ability of U1A to inhibit poly(A) polymerase correlate with the accessibility of the novel sites. However, long range interactions are responsible for maintaining them in an open configuration. Mutation of an RNase V1-sensitive site 102 nucleotides upstream, directly adjacent to the competing 5 splice site, changes the structure of one the U1A binding sites and thus abolishes the binding of the second U1A molecule and the ability of U1A ability to inhibit poly(A) polymerase activity at this site. These sites bind U1A via its N-terminal domain but with a 10-fold lower affinity than U1 small nuclear RNA. This lower binding affinity is more conducive to U1A's regulation of poly(A) tail addition to heterologous mRNA.The major source of diversity in metazoans is the ability to process pre-mRNA into alternative mRNAs via the selection of alternative splice sites and alternative 3Ј-ends, giving rise to a range of proteins encoded by the same gene that are differentially expressed during growth and development (for review see Ref.

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“…49 Determination of the U1 snRNA cleavage site via nuclease protection Nuclease S1 mapping was performed as described before 50 with some minor modifications. U1 snRNA (supplemented with 100 mg yeast RNA) and yeast RNA (as control) were annealed with 2.5610 4 c.p.m.…”

Section: Pcp Labelingmentioning

confidence: 99%

The fate of U1 snRNP during anti-Fas induced apoptosis: specific cleavage of the U1 snRNA molecule

et al. 2000

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During apoptosis, the U1-70K protein, a component of the spliceosomal U1 snRNP complex, is specifically cleaved by the enzyme caspase-3, converting it into a C-terminally truncated 40-kDa fragment. In this study, we show that the 40-kDa U1-70K fragment is still associated with the complete U1 snRNP complex, and that no obvious modifications occur with the U1 snRNP associated proteins U1A, U1C and Sm-B/B'. Furthermore, it is described for the first time that the U1 snRNA molecule, which is the backbone of the U1 snRNP complex, is modified during apoptosis by the specific removal of the first 5 ± 6 nucleotides including the 2,2,7-trimethylguanosine (TMG) cap. The observations that U1 snRNA cleavage is very specific (no such modifications were detected for the other U snRNAs tested) and that U1 snRNA cleavage is markedly inhibited in the presence of caspase inhibitors, indicate that an apoptotically activated ribonuclease is responsible for the specific modification of U1 snRNA during apoptosis. Cell Death and Differentiation (2000) 7, 70 ± 79.

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The human U1 snRNP-Specific U1A protein inhibits polyadenylation of its own pre-mRNA

Cited by 202 publications

References 60 publications

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

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

Sequences Adjacent to the 5′ Splice Site Control U1A Binding Upstream of the IgM Heavy Chain Secretory Poly(A) Site

The fate of U1 snRNP during anti-Fas induced apoptosis: specific cleavage of the U1 snRNA molecule

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