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

Santos, K.F.; Mozaffari‐Jovin, Sina; Weber, Gert; Peña, Vladimir; Lührmann, Reinhard; Wahl, Markus C.

doi:10.1073/pnas.1208098109

Cited by 93 publications

(219 citation statements)

References 26 publications

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“…Our observation that the isolated ILS dissociates completely in the presence of any rNTP was therefore very surprising in that it indicated that the ATP-specific Brr2 helicase/ ATPase is not required for the ILS disassembly. The recently determined X-ray crystal structure of Brr2 reveals a configuration of amino acid side chains at the nucleotide-binding pocket that exclusively allows binding of ATP (Santos et al 2012). It can thus be ruled out that Brr2 loses its ATP specificity in the context of the spliceosome.…”

Section: Discussionmentioning

confidence: 99%

Dissection of the factor requirements for spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system

et al. 2013

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The spliceosome is a single-turnover enzyme that needs to be dismantled after catalysis to both release the mRNA and recycle small nuclear ribonucleoproteins (snRNPs) for subsequent rounds of pre-mRNA splicing. The RNP remodeling events occurring during spliceosome disassembly are poorly understood, and the composition of the released snRNPs are only roughly known. Using purified components in vitro, we generated post-catalytic spliceosomes that can be dissociated into mRNA and the intron-lariat spliceosome (ILS) by addition of the RNA helicase Prp22 plus ATP and without requiring the step 2 proteins Slu7 and Prp18. Incubation of the isolated ILS with the RNA helicase Prp43 plus Ntr1/Ntr2 and ATP generates defined spliceosomal dissociation products: the intron-lariat, U6 snRNA, a 20-25S U2 snRNP containing SF3a/b, an 18S U5 snRNP, and the ''nineteen complex'' associated with both the released U2 snRNP and intron-lariat RNA. Our system reproduces the entire ordered disassembly phase of the spliceosome with purified components, which defines the minimum set of agents required for this process. It enabled us to characterize the proteins of the ILS by mass spectrometry and identify the ATPase action of Prp43 as necessary and sufficient for dissociation of the ILS without the involvement of Brr2 ATPase.[Keywords: intron-lariat spliceosome; disassembly; Prp22; Prp43; Brr2] Supplemental material is available for this article. Pre-mRNA splicing proceeds by way of two phosphoester transfer reactions and is catalyzed by the spliceosome, which consists of the U1, U2, U4/U6, and U5 small nuclear ribonucleoproteins (snRNPs) and numerous nonsnRNP proteins (Will and Lü hrmann 2011). The snRNPs are involved in recognizing short conserved sequences of the pre-mRNA, including the 59 and 39 splice sites (SS) and the branch site (BS), and in positioning the reactive nucleotides for catalysis. The spliceosome is a dynamic molecular machine, undergoing several major structural rearrangements during its functional cycle. These events are mediated by at least eight conserved DExD/H-box NTPases (hereafter termed RNA helicases) that act at discrete stages of the splicing pathway (Staley and Guthrie 1998;Cordin et al. 2012).Spliceosome assembly is initiated by the binding of U1 and U2 snRNPs to the 59 and the BS, respectively. This is followed by the recruitment of the U4/U6.U5 tri-snRNP, forming the precatalytic B complex. Catalytic activation of the spliceosome (leading to complex B act ) involves the displacement of U1 and U4 snRNAs from the spliceosome and the formation of new base pair interactions between the U6 and U2 snRNAs and the 59 SS (Staley and Guthrie 1998). The resulting RNA structure plays a central role in catalyzing both steps of splicing (Nilsen 1998).These RNA-RNA rearrangements are remodeled by the combined actions of the RNA helicases Prp28 and Brr2, which disrupt the base-pairing between U1 snRNA and the 59 SS and between the U4 and U6 snRNAs, respectively (Laggerbauer et al. 1998;Raghunathan and Guthrie 1998;Staley...

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Section: Discussionmentioning

confidence: 99%

Dissection of the factor requirements for spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system

et al. 2013

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“…(D) Interactions involving the E890 residue of human Brr2 (equivalent to E909 in yeast Brr2) in a crystal structure with ADP bound at the NC (PDB ID 4F93). 51 Dashed lines, hydrogen bonds or salt bridges. The rotation symbol indicates the orientation relative to (C).…”

Section: Brr2 Exhibits a Unique Structurementioning

confidence: 99%

“…2A,B). 41,52 Only the N-terminal helicase cassette (NC) is active in nucleotide hydrolysis and RNA unwinding, 51 and the enzymatic activity of the NC alone is required for splicing in vivo. 27 The C-terminal cassette (CC) is still able to bind ATP, 51 but it contains noncanonical residues in the ATP binding and hydrolysis motifs, which abrogate the ATPase activity.…”

Section: Brr2 Exhibits a Unique Structurementioning

confidence: 99%

“…41,52 Only the N-terminal helicase cassette (NC) is active in nucleotide hydrolysis and RNA unwinding, 51 and the enzymatic activity of the NC alone is required for splicing in vivo. 27 The C-terminal cassette (CC) is still able to bind ATP, 51 but it contains noncanonical residues in the ATP binding and hydrolysis motifs, which abrogate the ATPase activity. 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.…”

Section: Brr2 Exhibits a Unique Structurementioning

confidence: 99%

“…51 The Sec63 homology units consist of a helical bundle (HB), a helix-loop-helix (HLH) and an immunoglobulin-like (IG) domain ( 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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Spliceosome

Anthony

Krummel

2013

Encyclopedia of Life Sciences

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The removal of noncoding regions or introns and the splicing together of the protein‐coding regions or exons from precursor messenger ribonucleic acid (pre‐mRNA) transcripts is fundamental to metzoan cell development, as well as its maintenance. The nuclear process of pre‐mRNA splicing is a complex phenomenon catalysed by the ‘spliceosome’. The spliceosome consists of greater than a hundred protein and RNA molecules. Integral to the spliceosome are five RNA–protein complexes, the U1, U2, U4, U5 and U6 small nuclear ribonucleoproteins (snRNPs). The numerous proteins and the U snRNPs that make up the spliceosome come on and off during the spliceosome's reaction cycle. This article places an emphasis on our current understanding of the dynamics, composition and structure of the spliceosome. Key Concepts: Specific sequences defining the boundaries between a protein‐coding gene's noncoding regions or introns and its protein‐coding regions or exons are recognised by components of the pre‐mRNA splicing ‘machinery’ – the spliceosome. The spliceosome is composed of a large number of RNA and protein molecules. The protein subunits have diverse domain structures, whereas the RNA subunits form hydrogen bonding or base‐pairing interactions with critical sequences in a pre‐mRNA as well as with each other. The spliceosome reaction cycle involves an ordered assembly onto a pre‐mRNA transcript to ultimately catalyse two trans ‐esterification reactions that result in the removal or excision of an intron and the splicing together of two exons. Integral to the function of the spliceosome are five U snRNPs, each of which has a single RNA and 3–12 protein constituents including a homologous set of proteins (Sm or LSm) that forms a structural unit critical to the biogensis and integrity of each U snRNP. The U1, U2, U4 and U5 snRNPs all have a common core structure that consists of seven Sm proteins which assemble around a short U‐rich single‐stranded RNA sequence (the Sm site) present in the U1, U2, U4 and U5 snRNAs to form a heptameric ring with the single‐stranded RNA Sm site leafing through the central hole. Metazoan U1 snRNP functions to initiate the assembly of the spliceosome onto a pre‐mRNA transcript; its single RNA subunit (U1 snRNA) forms a cruciform‐like structure onto which the Sm proteins assemble to form the Sm core, while its three additional U‐specific proteins (U1‐A, U1‐70K, and U1‐C) participate in particle specific functions – including recognition of a pre‐mRNA transcripts 5' splice site sequence.

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

Cited by 93 publications

References 26 publications

Dissection of the factor requirements for spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system

Dissection of the factor requirements for spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system

Functions and regulation of the Brr2 RNA helicase during splicing

Spliceosome

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