Substrate-dependent Differences in U2AF Requirement for Splicing inAdenovirus-infected CellExtracts

Lützelberger, Martin; Backström, Ellenor; Akusjärvi, Göran

doi:10.1074/jbc.m413737200

Cited by 14 publications

(23 citation statements)

References 32 publications

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“…Also, our previous results have suggested that IIIa splicing is actively repressed in early infected cells by SR proteins binding to the 3RE (10), a repression that is relieved at the late phase of infection by a virus-induced dephosphorylation of SR proteins (23,24). Furthermore, the 3VDE, which binds U2AF inefficiently (11,12), adds to the low activity of IIIa splicing at early times of infection.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…Thus, we have shown that the 52,55K 3Ј splice site, which has an extended pyrimidine tract, requires U2AF 65 for activity (12). In contrast, the IIIa 3Ј splice site, which binds U2AF inefficiently (17), appears to be spliced by an U2AF-independent pathway (11,12). Thus, in the context of the L1 unit, two alternative pathways for 3Ј splice site recognition coexist: 52,55K splicing being activated by the normal U2AF pathway and IIIa splicing activated by the L4-33K protein-induced 3VDF pathway.…”

Section: Discussionmentioning

confidence: 99%

“…Standard splicing reaction mixtures (12,13) were incubated at 30°C for 60 -120 min in a total volume of 25 l with or without 0.75-30 pmol of a recombinant His-tagged L4-33K protein. Splicing products were resolved on 8% denaturing polyacrylamide gels.…”

Section: Methodsmentioning

confidence: 99%

“…The 3VDE consists of the IIIa branch point sequence, pyrimidine tract, and AG dinucleotide. Current results have suggested that the splicing enhancer activity of the 3VDE may operate through an U2AF-independent mechanism (11,12). In our working hypothesis we have proposed that a novel factor, of viral and/or cellular origin, the 3VDE interacting factor (3VDF), replaces U2AF as the pyrimidine tract binding factor in late-infected cells (9).…”

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confidence: 99%

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L4-33K, an Adenovirus-encoded Alternative RNA Splicing Factor

Törmänen¹,

Backström²,

Carlsson

et al. 2006

Journal of Biological Chemistry

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Splicing of the adenovirus IIIa mRNA is subjected to a strict temporal regulation during virus infection such that efficient IIIa 3 splice site usage is confined to the late phase of the infectious cycle. Here we show that the adenovirus L4-33K protein functions as a virus-encoded RNA splicing factor that preferentially activates splicing of transcripts with a weak 3 splice site sequence context, a sequence configuration that is shared by many of the late adenovirus 3 splice sites. Furthermore, we show that L4-33K activates IIIa splicing through the IIIa virus infection-dependent splicing enhancer element (3VDE). This element was previously shown to be the minimal element, both necessary and sufficient, for activation of IIIa splicing in the context of an adenovirus-infected cell. L4-33K stimulates an early step in spliceosome assembly and appears to be the only viral protein necessary to convert a nuclear extract prepared from uninfected HeLa cells to an extract with splicing properties very similar to a nuclear extract prepared from adenovirus lateinfected cells. Collectively, our results suggest that L4-33K is the key viral protein required to activate the early to late switch in adenovirus major late L1 alternative splicing.Most late adenovirus proteins are translated from mRNAs originating from the major late transcription unit (MLTU), 3 which extends from the major late promoter (MLP) at coordinate 16.8 to a termination signal close to the right-hand end of the genome (reviewed in Ref. 1). The ϳ28,000-nucleotide pre-mRNA expressed from the MLTU becomes polyadenylated at one of five possible sites, generating five families of mRNAs with co-terminal 3Ј-ends (L1-L5; Fig. 1A). Following selection of a poly(A) site, the primary transcript is spliced in such a way that each mature mRNA receives a common set of three short 5Ј-leader segments, the tripartite leader (Fig. 1A). This leader is then spliced to one of several alternative 3Ј splice sites, generating a total of more than 20 cytoplasmic mRNAs.The accumulation of mRNA from the MLTU is subjected to a temporal regulation at the levels of transcription elongation, poly(A) site choice and alternative 3Ј splice site selection (reviewed in Ref. 1). Thus, during the early phase of infection the MLP is active at a level comparable with the other early transcription units, whereas the same promoter accounts for most of the transcriptional activity at late times of infection (2, 3). However, at early times transcription initiated at the MLP decreases gradually over a large region beginning after the L1 unit, with few RNA polymerases extending beyond the L3 polyadenylation sequence (4). At late times, this block in elongation is alleviated and transcripts initiated at the MLP continue to the right hand end of the genome. The control of MLTU transcription is further regulated by events taking place at the level of poly(A) and alternative 3Ј splice site selection (2, 5, 6). Thus, although nuclear transcription proceeds across at least the L1, L2 and L3 poly(A) sites a...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

L4-33K, an Adenovirus-encoded Alternative RNA Splicing Factor

Törmänen¹,

Backström²,

Carlsson

et al. 2006

Journal of Biological Chemistry

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Formation of adenovirus DNA replication compartments

Hidalgo

González

2019

FEBS Letters

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Adenoviruses induce an extensive reorganization of the host cell nucleus during replication. Such a process results in the assembly of viral and cellular macromolecules into nuclear structures called adenovirus replication compartments (AdRCs), which function as platforms for viral DNA replication and gene expression. AdRCs co-opt host proteins and cellular pathways that restrict viral replication, suggesting that the mechanisms that control AdRC formation and function are essential for viral replication and lay at the basis of virus-host interactions. Here, we review the hallmarks of AdRCs and recent progress in our understanding of the formation, composition, and function of AdRCs. Furthermore, we discuss how AdRCs facilitate the interplay between viral and cellular machineries and hijack cellular functions to promote viral genome replication and expression.

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Allele-specific recognition of the 3′ splice site of INS intron 1

Královičová

Vořechovský

2010

Hum Genet

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Genetic predisposition to type 1 diabetes (T1D) has been associated with a chromosome 11 locus centered on the proinsulin gene (INS) and with differential steady-state levels of INS RNA from T1D-predisposing and -protective haplotypes. Here, we show that the haplotype-specific expression is determined by INS variants that control the splicing efficiency of intron 1. The adenine allele at IVS1-6 (rs689), which rapidly expanded in modern humans, renders the 3′ splice site of this intron more dependent on the auxiliary factor of U2 small nuclear ribonucleoprotein (U2AF). This interaction required both zinc fingers of the 35-kD U2AF subunit (U2AF35) and was associated with repression of a competing 3′ splice site in INS exon 2. Systematic mutagenesis of reporter constructs showed that intron 1 removal was facilitated by conserved guanosine-rich enhancers and identified additional splicing regulatory motifs in exon 2. Sequencing of intron 1 in primates revealed that relaxation of its 3′ splice site in Hominidae coevolved with the introduction of a short upstream open reading frame, providing a more efficient coupled splicing and translation control. Depletion of SR proteins 9G8 and transformer-2 by RNA interference was associated with exon 2 skipping whereas depletion of SRp20 with increased representation of transcripts containing a cryptic 3′ splice site in the last exon. Together, these findings reveal critical interactions underlying the allele-dependent INS expression and INS-mediated risk of T1D and suggest that the increased requirement for U2AF35 in higher primates may hinder thymic presentation of autoantigens encoded by transcripts with weak 3′ splice sites.Electronic supplementary materialThe online version of this article (doi:10.1007/s00439-010-0860-1) contains supplementary material, which is available to authorized users.

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Substrate-dependent Differences in U2AF Requirement for Splicing inAdenovirus-infected CellExtracts

Cited by 14 publications

References 32 publications

L4-33K, an Adenovirus-encoded Alternative RNA Splicing Factor

L4-33K, an Adenovirus-encoded Alternative RNA Splicing Factor

Formation of adenovirus DNA replication compartments

Allele-specific recognition of the 3′ splice site of INS intron 1

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