Inhibition of RNA Helicase Brr2 by the C-Terminal Tail of the Spliceosomal Protein Prp8

Mozaffari‐Jovin, Sina; Wandersleben, Traudy; Santos, K.F.; Will, Cindy L.; Lührmann, Reinhard; Wahl, Markus C.

doi:10.1126/science.1237515

Cited by 126 publications

(229 citation statements)

References 29 publications

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“…53,56 The functional relevance of this mode of Brr2 inhibition is underscored by the observation that mutations in Prp8, which affect residues in the Jab1 C-terminal tail and interfere with its function, lead to a severe form of retinitis pigmentosa in human. 53,88 The tail insertion was first observed in the structure of a human Brr2-Jab1 complex, 53 in which the Brr2 subunit lacked all elements of the NTR except the NC-clamp, but was not seen in a slightly further truncated yeast Brr2-Jab1 complex. 38 These findings raised the question whether the Jab1 tail insertion is conserved among organisms.…”

Section: Brr2 Regulation Via Trans-acting Factorsmentioning

confidence: 99%

“…Layer II -the CC as an intra-molecular helicase cofactor Close contacts between the active NC and inactive CC in crystal structures of Brr2 38,51,53 suggest that the NC and CC together form a larger functional unit. Indeed, deletion of the entire CC, exchange of residues in a long, irregularly structured linker that connects the cassettes, or exchange of residues that mediate NC-CC contacts affect the ATPase and helicase activities of the NC.…”

Section: Multiple Layers Of Helicase-associated Domains In Brr2mentioning

confidence: 99%

“…38,53 Thus, the first layer of helicase-associated domains in Brr2 directly modulate the enzymatic mechanism of the enzyme and contribute to its regulation by trans-acting factors.…”

Section: Multiple Layers Of Helicase-associated Domains In Brr2mentioning

confidence: 99%

“…2B). 38,53 In this position, it can insert an intrinsically disordered C-terminal tail into the Brr2 RNA-binding tunnel, thereby inhibiting accommodation of a substrate strand and consequently RNA duplex unwinding (Fig. 2B).…”

Section: Brr2 Regulation Via Trans-acting Factorsmentioning

confidence: 99%

“…27,51 Crystal structures of Brr2 helicase regions confirmed a similar globular architecture of the 2 helicase cassettes and revealed that the cassettes can intimately interact with each other via a large interface. 38,51,53 Preceding its 2 helicase cassettes, Brr2 contains a »450-residue N-terminal region (NTR; Fig. 2A,B).…”

Section: Brr2 Exhibits a Unique Structurementioning

confidence: 99%

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Functions and regulation of the Brr2 RNA helicase during splicing

2016

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Pre-mRNA splicing entails the stepwise assembly of an inactive spliceosome, its catalytic activation, splicing catalysis and spliceosome disassembly. Transitions in this reaction cycle are accompanied by compositional and conformational rearrangements of the underlying RNA-protein interaction networks, which are driven and controlled by 8 conserved superfamily 2 RNA helicases. The Ski2-like helicase, Brr2, provides the key remodeling activity during spliceosome activation and is additionally implicated in the catalytic and disassembly phases of splicing, indicating that Brr2 needs to be tightly regulated during splicing. Recent structural and functional analyses have begun to unravel how Brr2 regulation is established via multiple layers of intra-and inter-molecular mechanisms. Brr2 has an unusual structure, including a long N-terminal region and a catalytically inactive C-terminal helicase cassette, which can auto-inhibit and auto-activate the enzyme, respectively. Both elements are essential, also serve as protein-protein interaction devices and the N-terminal region is required for stable Brr2 association with the tri-snRNP, tri-snRNP stability and retention of U5 and U6 snRNAs during spliceosome activation in vivo. Furthermore, a C-terminal region of the Prp8 protein, comprising consecutive RNase H-like and Jab1/MPN-like domains, can both up-and downregulate Brr2 activity. Biochemical studies revealed an intricate cross-talk among the various cis-and transregulatory mechanisms. Comparison of isolated Brr2 to electron cryo-microscopic structures of yeast and human U4/U6 U5 tri-snRNPs and spliceosomes indicates how some of the regulatory elements exert their functions during splicing. The various modulatory mechanisms acting on Brr2 might be exploited to enhance splicing fidelity and to regulate alternative splicing. KEYWORDSPre-mRNA splicing; regulation of pre-mRNA splicing; remodeling of RNAprotein complexes; RNA helicase structure, function and regulation; spliceosome catalytic activation

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Section: Brr2 Regulation Via Trans-acting Factorsmentioning

confidence: 99%

Section: Multiple Layers Of Helicase-associated Domains In Brr2mentioning

confidence: 99%

“…38,53 Thus, the first layer of helicase-associated domains in Brr2 directly modulate the enzymatic mechanism of the enzyme and contribute to its regulation by trans-acting factors.…”

Section: Multiple Layers Of Helicase-associated Domains In Brr2mentioning

confidence: 99%

Section: Brr2 Regulation Via Trans-acting Factorsmentioning

confidence: 99%

Section: Brr2 Exhibits a Unique Structurementioning

confidence: 99%

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Functions and regulation of the Brr2 RNA helicase during splicing

2016

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Inhibition of SNW1 association with spliceosomal proteins promotes apoptosis in breast cancer cells

et al. 2014

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RNA splicing is a fundamental process for protein synthesis. Recent studies have reported that drugs that inhibit splicing have cytotoxic effects on various tumor cell lines. In this report, we demonstrate that depletion of SNW1, a component of the spliceosome, induces apoptosis in breast cancer cells. Proteomics and biochemical analyses revealed that SNW1 directly associates with other spliceosome components, including EFTUD2 (Snu114) and SNRNP200 (Brr2). The SKIP region of SNW1 interacted with the N-terminus of EFTUD2 as well as two independent regions in the C-terminus of SNRNP200. Similar to SNW1 depletion, knockdown of EFTUD2 increased the numbers of apoptotic cells. Furthermore, we demonstrate that exogenous expression of either the SKIP region of SNW1 or the N-terminus region of EFTUD2 significantly promoted cellular apoptosis. Our results suggest that the inhibition of SNW1 or its associating proteins may be a novel therapeutic strategy for cancer treatment.

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Retinitis Pigmentosa Mutations ofSNRNP200Enhance Cryptic Splice-Site Recognition

2013

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Mutations in SNRP200 gene cause autosomal-dominant retinal disorder retinitis pigmentosa (RP). The protein product of SNRNP200 is BRR2, a DExD/H box RNA helicase crucial for pre-mRNA splicing. In this study, we prepared p.S1087L and p.R1090L mutations of human BRR2 using bacterial artificial chromosome recombineering and stably expressed them in human cell culture. Mutations in BRR2 did not compromise snRNP assembly and both mutants were incorporated into the spliceosome just as the wild-type (wt) protein. Surprisingly, cells expressing RP mutants exhibited increased splicing efficiency of the LDHA gene. Next, we found that depletion of endogenous BRR2 enhanced usage of a β-globin cryptic splice site while splicing at the correct splice site was inhibited. Proper splicing of optimal and cryptic splice sites was restored in cells expressing BRR2-wt but not in cells expressing RP mutants. Taken together, our data suggest that BRR2 is an important factor in 5'-splice-site recognition and that the RP-linked mutations c.3260C>T (p.S1087L) and c.3269G>T (p.R1090L) affect this BRR2 function.

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Inhibition of RNA Helicase Brr2 by the C-Terminal Tail of the Spliceosomal Protein Prp8

Cited by 126 publications

References 29 publications

Functions and regulation of the Brr2 RNA helicase during splicing

Functions and regulation of the Brr2 RNA helicase during splicing

Inhibition of SNW1 association with spliceosomal proteins promotes apoptosis in breast cancer cells

Retinitis Pigmentosa Mutations ofSNRNP200Enhance Cryptic Splice-Site Recognition

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