The network of protein–protein interactions within the human U4/U6.U5 tri-snRNP

Liu, Sunbin; Rauhut, Reinhard; Vornlocher, Hans‐Peter; Lührmann, Reinhard

doi:10.1261/rna.55406

Cited by 172 publications

(230 citation statements)

References 57 publications

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“…hPrp3p presumably plays an important role in recruiting other splicing factors like hPrp4p to the U4/U6 small ribonucleoprotein and docking the U4/U6/U5 tri-snRNP to the prespliceosome (Liu et al, 2006). In the present study we describe that hPrp3p interacts with protein kinase CK2 in vitro and in vivo and that is phosphorylated by this kinase.…”

Section: Introductionmentioning

confidence: 52%

Protein kinase CK2 interacts with the splicing factor hPrp3p

et al. 2007

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Numerous signalling pathways in cells are influenced by the ubiquitous Ser/Thr protein kinase CK2. Protein kinase CK2 is composed of two regulatory b-subunits and two catalytic a-or a 0 -subunits. Several of the known CK2 substrates are proteins known to regulate transcriptional events. Here, we describe that protein kinase CK2 interacts with the splicing factor hPrp3p, which is important for the assembly of the spliceosome. In a twohybrid screen hPrp3p is exclusively bound to the catalytic a-or a 0 -subunits of CK2 but not to the regulatory bsubunit. The interaction was confirmed by coimmunoprecipitation experiments in vitro and in vivo. Moreover, both proteins colocalized in nuclear speckles which is typical for splicing factor compartments within the nucleus. Phosphorylation experiments revealed that hPrp3p is also a substrate of protein kinase CK2. The main phosphorylation site was mapped to C-terminal residues. In vitro and in vivo splicing assays showed that the splicing activity is significantly influenced by the CK2-hPrp3p interaction. Thus, these data showed that CK2 is involved in the regulation of RNA processing.

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Section: Introductionmentioning

confidence: 52%

Protein kinase CK2 interacts with the splicing factor hPrp3p

et al. 2007

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“…A number of proteins essential for different steps of splicing interact with the C-terminal cassette of Brr2 (24,25). The ability of the C-terminal cassette to transmit signals to the N-terminal cassette suggests that these proteins may not merely use the C-terminal cassette as a passive landing pad but also to influence the N-terminal cassette from a distance.…”

Section: Discussionmentioning

confidence: 99%

Structural basis for functional cooperation between tandem helicase cassettes in Brr2-mediated remodeling of the spliceosome

Santos

Mozaffari‐Jovin

Weber

et al. 2012

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

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Assembly of a spliceosome, catalyzing precursor-messenger RNA splicing, involves multiple RNA-protein remodeling steps, driven by eight conserved DEXD/H-box RNA helicases. The 250-kDa Brr2 enzyme, which is essential for U4/U6 di-small nuclear ribonucleoprotein disruption during spliceosome catalytic activation and for spliceosome disassembly, is the only member of this group that is permanently associated with the spliceosome, thus requiring its faithful regulation. At the same time, Brr2 represents a unique subclass of superfamily 2 nucleic acid helicases, containing tandem helicase cassettes. Presently, the mechanistic and regulatory consequences of this unconventional architecture are unknown. Here we show that in human Brr2, two ring-like helicase cassettes intimately interact and functionally cooperate and how retinitis pigmentosa-linked Brr2 mutations interfere with the enzyme's function. Only the N-terminal cassette harbors ATPase and helicase activities in isolation. Comparison with other helicases and mutational analyses show how it threads single-stranded RNA, and structural features suggest how it can load onto an internal region of U4/U6 di-snRNA. Although the C-terminal cassette does not seem to engage RNA in the same fashion, it binds ATP and strongly stimulates the N-terminal helicase. Mutations at the cassette interface, in an intercassette linker or in the C-terminal ATP pocket, affect this cross-talk in diverse ways. Together, our results reveal the structural and functional interplay between two helicase cassettes in a tandem superfamily 2 enzyme and point to several sites through which Brr2 activity may be regulated.pre-mRNA splicing | RNA helicase Brr2 | X-ray crystallography N ucleotide triphosphate-dependent nucleic acid unwindases ("helicases") serve as motors and regulators of many biological macromolecular machines. Assembly of a spliceosome, catalyzing precursor-messenger RNA splicing, is a paradigmatic case that involves multiple RNA-protein remodeling steps, driven by eight conserved RNA helicases of the DEXD/H-box family (1). None of the spliceosome's small nuclear ribonucleoprotein (snRNP) subunits (U1, U2, U4, U5, and U6 in the major spliceosome) or its plethora of non-snRNP factors bear a preformed active center for splicing catalysis. Instead, profound compositional and conformational changes are required to convert an initial, inactive assembly to a catalytically competent spliceosome (2).Catalytic activation involves the unwinding of the U4 and U6 snRNAs, which are extensively base-paired via two regions (stems 1 and 2) when delivered to the spliceosome in the framework of the U4/U6-U5 tri-snRNP. As the U5 snRNP protein, Brr2, unwinds U4/U6 duplexes in vitro (3, 4) and Brr2 mutations interfere with catalytic activation (5-7), the enzyme is thought to elicit these rearrangements. Brr2 already encounters its U4/U6 substrate in the U4/U6-U5 tri-snRNP, but U4/U6 dissociation must be delayed until splice sites have been reliably located during spliceosome assembly. Furthermore, unl...

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“…Early reports speculated that HAT repeats might bind RNA (1,5,7). However, a lack of evidence for RNA binding and the fact that some HAT repeats can bind peptides (8,9) have led recent literature to assume that HAT motifs serve only to support protein-protein interactions (3, 9, 10). Our demonstration that the HAT protein HCF107 binds RNA with high affinity and sequence specificity together with the exclusive association of HAT proteins with RNA-containing complexes strongly suggest that RNA binding activity contributes to the biological functions of most HAT domains.…”

Section: Discussionmentioning

confidence: 99%

“…Crystal structures of CstF-77 confirmed that HAT repeat tracts adopt a TPR-like structure (3, 4). The possibility that HAT repeat tracts might bind RNA has been suggested (1, 5-7), but the notion that HAT repeat tracts serve as a scaffold for binding other proteins dominates recent literature (3,(8)(9)(10)). This view is reflected by the annotation of the HAT motif at InterPro, which states only that "the repeats may be involved in protein-protein interactions" (http://www.ebi.…”

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confidence: 99%

RNA binding and RNA remodeling activities of the half-a-tetratricopeptide (HAT) protein HCF107 underlie its effects on gene expression

Hammani

Cook

Barkan

2012

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

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The half-a-tetratricopeptide repeat (HAT) motif is a helical repeat motif found in proteins that influence various aspects of RNA metabolism, including rRNA biogenesis, RNA splicing, and polyadenylation. This functional association with RNA suggested that HAT repeat tracts might bind RNA. However, RNA binding activity has not been reported for any HAT repeat tract, and recent literature has emphasized a protein binding role. In this study, we show that a chloroplast-localized HAT protein, HCF107, is a sequence-specific RNA binding protein. HCF107 consists of 11 tandem HAT repeats and short flanking regions that are also predicted to form helical hairpins. The minimal HCF107 binding site spans ∼11 nt, consistent with the possibility that HAT repeats bind RNA through a modular one repeat-1 nt mechanism. Binding of HCF107 to its native RNA ligand in the psbH 5′ UTR remodels local RNA structure and protects the adjacent RNA from exonucleases in vitro. These activities can account for the RNA stabilizing, RNA processing, and translational activation functions attributed to HCF107 based on genetic data. We suggest that analogous activities contribute to the functions of HAT domains found in ribonucleoprotein complexes in the nuclear-cytosolic compartment.helical repeat protein | plastid | pentatricopeptide repeat T he half-a-tetratricopeptide (HAT) motif is a helical repeat motif that has been functionally linked to RNA because of its presence exclusively in complexes that influence RNA metabolism (1). Examples of HAT domain proteins include Utp6, which is involved in nuclear pre-rRNA processing, Prp6, which is involved in nuclear pre-mRNA splicing, and CstF-77, which is involved in pre-mRNA cleavage and polyadenylation. The HAT motif is related to the tetratricopeptide repeat (TPR), a degenerate 34-aa motif that forms a pair of antiparallel α-helices (reviewed in ref.2). TPR motifs are typically found in tandem arrays, which stack to form a broad surface that binds protein ligands. Crystal structures of CstF-77 confirmed that HAT repeat tracts adopt a TPR-like structure (3, 4). The possibility that HAT repeat tracts might bind RNA has been suggested (1, 5-7), but the notion that HAT repeat tracts serve as a scaffold for binding other proteins dominates recent literature (3,(8)(9)(10)). This view is reflected by the annotation of the HAT motif at InterPro, which states only that "the repeats may be involved in protein-protein interactions" (http://www.ebi. ac.uk/interpro/IEntry?ac=IPR003107).Although TPR, HEAT, and several other helical repeat motifs form protein binding surfaces, similar structures have been shown to bind nucleic acids. Puf and pentatricopeptide repeat (PPR) motifs have been shown to bind RNA (reviewed in refs. 11 and 12), and mTERF, ALK, and TAL repeat motifs have been shown to bind DNA (reviewed in refs. 13 and 14). Thus, it seemed quite plausible that the presence of HAT domains exclusively in ribonucleoprotein complexes reflects the fact that HAT repeat tracts interact directly with RNA. In this ...

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The network of protein–protein interactions within the human U4/U6.U5 tri-snRNP

Cited by 172 publications

References 57 publications

Protein kinase CK2 interacts with the splicing factor hPrp3p

Protein kinase CK2 interacts with the splicing factor hPrp3p

Structural basis for functional cooperation between tandem helicase cassettes in Brr2-mediated remodeling of the spliceosome

RNA binding and RNA remodeling activities of the half-a-tetratricopeptide (HAT) protein HCF107 underlie its effects on gene expression

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