A composite double-/single-stranded RNA-binding region in protein Prp3 supports tri-snRNP stability and splicing

Liu, Sunbin; Mozaffari‐Jovin, Sina; Wollenhaupt, J.; Santos, K.F.; Theuser, Matthias; Dunin-Horkawicz, Stanisław; Fabrizio, Patrizia; Bujnicki, Janusz M.; Lührmann, Reinhard; Wahl, Markus C.

doi:10.7554/elife.07320

Cited by 31 publications

(38 citation statements)

References 75 publications

(99 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This is consistent with a recent crystal structure of Prp3 in association with portions of U4/U6. 20 We compared the CmSnu13-RNA interaction 5 with reported observations for Snu13 orthologs. The dissociation constant, K D , for for CmSnu13 binding U4 snRNA was measured using fluorescence polarization with a 5'-fluorescein labeled RNA corresponding to U4 stem II, 5 nucleotides 22-50 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…11,[13][14][15][16][17][18] It has also been shown that Prp3p (human 90K) and Prp4p (human 60K) associate and require Snu13p to bind to U4 snRNA prior to their own assembly into the U4 snRNP. 14,20 It is not known what proteins are associated with U4 snRNA prior to assembly of the U4/U6 di-snRNP, but they may include the core Sm and Snu13 proteins. 14,21 In the second stage of U4/U6 snRNP assembly, Prp31 and Prp3/4 join independently.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Conserved structure of Snu13 from the highly reduced spliceosome of Cyanidioschyzon merolae

et al. 2016

View full text Add to dashboard Cite

Structural and functional analysis of proteins involved in pre-mRNA splicing is challenging because of the complexity of the splicing machinery, known as the spliceosome. Bioinformatic, proteomic, and biochemical analyses have identified a minimal spliceosome in the red alga Cyanidioschyzon merolae. This spliceosome consists of only 40 core proteins, compared to~70 in S. cerevisiae (yeast) and~150 in humans. We report the X-ray crystallographic analysis of C. merolae Snu13 (CmSnu13), a key component of the assembling spliceosome, and present evidence for conservation of Snu13 function in this algal splicing pathway. The near identity of CmSnu13's threedimensional structure to yeast and human Snu13 suggests that C. merolae should be an excellent model system for investigating the structure and function of the conserved core of the spliceosome.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Conserved structure of Snu13 from the highly reduced spliceosome of Cyanidioschyzon merolae

et al. 2016

View full text Add to dashboard Cite

show abstract

“…of the U4/U6 duplex, formed by stem I, stem II, and an intervening U4 5′ stem loop (5′SL) (22,(29)(30)(31) (Fig. 1A).…”

mentioning

confidence: 99%

“…They bind U4/U6 di-snRNA in a hierarchical manner, with Snu13 required for Prp31 binding (29,32); both proteins together enhance binding of Prp3 (30,32). Snu13 and Prp31 bind the U4 5′SL and maintain additional contacts to stems I and II, whereas Prp3 binds stem II and a U6 3′-overhang (22,(29)(30)(31)(32)(33). However, it is presently not known how U4/U6-bound proteins influence Brr2-mediated U4/U6 unwinding, which regions of the U4/U6 di-snRNA Brr2 actively unwind, and how U4/U6 components segregate upon Brr2-mediated U4/U6 di-snRNP disruption.…”

mentioning

confidence: 99%

Substrate-assisted mechanism of RNP disruption by the spliceosomal Brr2 RNA helicase

Theuser

Höbartner

Wahl

et al. 2016

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

Self Cite

View full text Add to dashboard Cite

The Brr2 RNA helicase disrupts the U4/U6 di-small nuclear RNAprotein complex (di-snRNP) during spliceosome activation via ATPdriven translocation on the U4 snRNA strand. However, it is unclear how bound proteins influence U4/U6 unwinding, which regions of the U4/U6 duplex the helicase actively unwinds, and whether U4/U6 components are released as individual molecules or as subcomplexes. Here, we set up a recombinant Brr2-mediated U4/U6 disnRNP disruption system, showing that sequential addition of the U4/U6 proteins small nuclear ribonucleoprotein-associated protein 1 (Snu13), pre-mRNA processing factor 31 (Prp31), and Prp3 to U4/U6 di-snRNA leads to a stepwise decrease of Brr2-mediated U4/U6 unwinding, but that unwinding is largely restored by a Brr2 cofactor, the C-terminal Jab1/MPN domain of the Prp8 protein.Brr2-mediated U4/U6 unwinding was strongly inhibited by mutations in U4/U6 di-snRNAs that diminish the ability of U6 snRNA to adopt an alternative conformation but leave the number and kind of U4/U6 base pairs unchanged. Irrespective of the presence of the cofactor, the helicase segregated a Prp3-Prp31-Snu13-U4/U6 RNP into an intact Prp31-Snu13-U4 snRNA particle, free Prp3, and free U6 snRNA. Together, these observations suggest that Brr2 translocates only a limited distance on the U4 snRNA strand and does not actively release RNA-bound proteins. Unwinding is then completed by the partially displaced U6 snRNA adopting an alternative conformation, which leads to dismantling of the Prp3-binding site on U4/U6 di-snRNA but leaves the Prp31-and Snu13-binding sites on U4 snRNA unaffected. In this fashion, Brr2 can activate the spliceosome by stripping U6 snRNA of all precatalytic binding partners, while minimizing logistic requirements for U4/U6 di-snRNP reassembly after splicing.pre-mRNA splicing | RNA helicase | RNP remodeling | small nuclear ribonucleoprotein particle | spliceosome catalytic activation R NA helicases form a large family of nucleotide triphosphatedependent enzymes, many of which can unwind RNA duplexes in vitro (1, 2). However, in vivo, these enzymes typically act on RNA-protein complexes (RNPs) and can also exhibit other activities, including RNA annealing (3, 4), RNA clamping (5), displacement of RNA-bound proteins (6), or displacement of RNAbound RNPs (7). Often, the precise functions of RNA helicases are unclear because their in vivo substrates are mostly unknown. Consequently, the RNP remodeling activities of RNA helicases have typically been studied using model RNPs that these helicases would not encounter naturally (6, 7).Eight superfamily (SF) 2 RNA helicases are required for premRNA splicing by the spliceosome (8). During every splicing reaction, these enzymes drive and control multiple compositional and conformational RNP remodeling events that accompany the initial assembly of an inactive spliceosome, its catalytic activation, its splicing catalysis, and its disassembly (8, 9). The most profound helicase-mediated rearrangements occur during spliceosome activation. In the precatalyt...

show abstract

“…On the other hand, isolated Brr2 is a comparatively weak helicase, 25,40,41 and its U4/U6 di-snRNA substrate is stabilized by extensive base pairing and bound proteins, [12][13][14]29,42,43 suggesting that the helicase may also depend on specific activation to efficiently unwind the U4/U6 duplex at the right time.…”

Section: Brr2 Requires Tight Regulationmentioning

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

A composite double-/single-stranded RNA-binding region in protein Prp3 supports tri-snRNP stability and splicing

Cited by 31 publications

References 75 publications

Conserved structure of Snu13 from the highly reduced spliceosome of Cyanidioschyzon merolae

Conserved structure of Snu13 from the highly reduced spliceosome of Cyanidioschyzon merolae

Substrate-assisted mechanism of RNP disruption by the spliceosomal Brr2 RNA helicase

Functions and regulation of the Brr2 RNA helicase during splicing

Contact Info

Product

Resources

About