Release of SF3 from the intron branchpoint activates the first step of pre-mRNA splicing

Lardelli, Rea M.; Thompson, James; Yates, John R.; Stevens, Scott W.

doi:10.1261/rna.2030510

Cited by 156 publications

(218 citation statements)

References 76 publications

Supporting

Mentioning

194

Contrasting

Unclassified

Order By: Relevance

“…These include the U5, the NTC, most of the NTC-related, and all U2 proteins. The latter is surprising, as it has been reported that the U2 SF3a/b proteins dissociate from the spliceosome already at the catalytic stage of splicing (Lardelli et al 2010). It has been suggested that dissociation of U2 SF3a/b proteins might be necessary for catalysis (Lardelli et al 2010).…”

Section: Discussionmentioning

confidence: 99%

“…The transition from the B to the B act complex involves a significant change in the spliceosome's protein composition, with numerous polypeptides such as the U1 and U4/U6 proteins being displaced, while ;20 others, including those of the NTC (nineteen complex) are stably recruited to the B act complex (Chan et al 2003;Fabrizio et al 2009;Warkocki et al 2009). For catalytic activation of the spliceosome, the DEAH-box helicase Prp2 binds together with a cofactor, Spp2, and restructures the B act complex in an ATP-dependent manner, yielding the B* complex (Roy et al 1995;Silverman et al 2004); this leads to, among other things, destabilization and/or disruption of the binding of several spliceosomal proteins (Warkocki et al 2009;Lardelli et al 2010;Ohrt et al 2012). Upon recruitment of the step 1 factor Cwc25 to the B* complex, step 1 catalysis occurs, whereby the adenosine at the BS attacks the 59 SS, generating the cleaved exon 1 and intron-lariat (IL)-39 exon intermediates (Chiu et al 2009;Warkocki et al 2009).…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2013

View full text Add to dashboard Cite

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...

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

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

View full text Add to dashboard Cite

show abstract

“…1). Prp2 is required for the transition from the B act to the B Ã complex, 104 during which it remodels binding of the multimeric SF3b complex to the branch point region, 17,105 reduces association of SF3a and SF3b proteins, 106 leads to displacement of the Cwc24 and Cwc27 components of the 19 complex (NTC) as well as of the Bud13 subunit of the RNA retention and splicing (RES) complex 106 and proofreads the catalytic core of the spliceosome. 107 Consistent with some of these functions, the SF3B1 (Hsh155) protein in the B act complex bridges between the plug domain of Brr2 and Prp2.…”

Section: Brr2 Regulation During Splicingmentioning

confidence: 99%

Functions and regulation of the Brr2 RNA helicase during splicing

2016

View full text Add to dashboard Cite

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

show abstract

“…8 It forms a dynamic and integral part of 4 intermediary complexes (A, B, B Ã and B act ). Previous studies have revealed various roles for SF3b complex including i) recognition of branch point adenosine 9 and promotion of stable interaction for U2 and U11/U12 di-snRNP to pre-mRNA, 10 ii) prevention of premature splicing 10 and iii) interaction with snRNAs and pre-mRNA. 11,12 The complex is made of 7 proteins, namely, p14, SF3b49, SF3b145, SF3b155, SF3b10, SF3b130 and SF3b14b.…”

Section: Introductionmentioning

confidence: 99%

“…9,15 This enables SF3b to present a temporary steric barrier to branch point sequence (BPS) prior to activation. 10 In addition, SF3b49 has been shown to interact with the stemloops at 5 0 end of U2 snRNA 11 and is also shown to be cross linked around the BPS along with the other components such as the SF3b145 and SF3b155. 12 Further, SF3b49 and SF3b145 are known to participate in multiple protein-protein interactions during transition from A to C in the dynamic splicing pathway.…”

Section: Introductionmentioning

confidence: 99%

Structural and mechanistic insights into human splicing factor SF3b complex derived using an integrated approach guided by the cryo-EM density maps

et al. 2016

View full text Add to dashboard Cite

Pre-mRNA splicing in eukaryotes is performed by the spliceosome, a highly complex macromolecular machine. SF3b is a multi-protein complex which recognizes the branch point adenosine of pre-mRNA as part of a larger U2 snRNP or U11/U12 di-snRNP in the dynamic spliceosome machinery. Although a cryo-EM map is available for human SF3b complex, the structure and relative spatial arrangement of all components in the complex are not yet known. We have recognized folds of domains in various proteins in the assembly and generated comparative models. Using an integrative approach involving structural and other experimental data, guided by the available cryo-EM density map, we deciphered a pseudoatomic model of the closed form of SF3b which is found to be a "fuzzy complex" with highly flexible components and multiplicity of folds. Further, the model provides structural information for 5 proteins (SF3b10, SF3b155, SF3b145, SF3b130 and SF3b14b) and localization information for 4 proteins (SF3b10, SF3b145, SF3b130 and SF3b14b) in the assembly for the first time. Integration of this model with the available U11/U12 di-snRNP cryo-EM map enabled elucidation of an open form. This now provides new insights on the mechanistic features involved in the transition between closed and open forms pivoted by a hinge region in the SF3b155 protein that also harbors cancer causing mutations. Moreover, the open form guided model of the 5 0 end of U12 snRNA, which includes the branch point duplex, shows that the architecture of SF3b acts as a scaffold for U12 snRNA: pre-mRNA branch point duplex formation with potential implications for branch point adenosine recognition fidelity.

show abstract

Release of SF3 from the intron branchpoint activates the first step of pre-mRNA splicing

Cited by 156 publications

References 76 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

Structural and mechanistic insights into human splicing factor SF3b complex derived using an integrated approach guided by the cryo-EM density maps

Contact Info

Product

Resources

About