Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly

Didychuk, Allison L.; Eric, Montemayor,; Brow, David A.; Butcher, Samuel E.

doi:10.1093/nar/gkv1374

Cited by 24 publications

(52 citation statements)

References 46 publications

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“…1A). Indeed, telestem mutations that are expected to destabilize the ISL and induce an alternative U6 structure give rise to enhanced spontaneous U4/U6 annealing (36), and destabilization of the telestem in the human system promotes U4/U6 formation (37). Telestem formation is prevented in yeast U4/U6 (38), presumably by the coaxially stacked U4/U6 stems I and II intervening between its branches (22,31,39).…”

Section: Discussionmentioning

confidence: 99%

“…Prp24 may have a second function in disrupting U6/U2 interactions after splicing (48,49). During U4/U6 annealing, three RNA recognition motifs of Prp24 interlock with the U6 ISL, asymmetrical bulge, and telestem (48), giving rise to an electropositive groove, which could mediate U4/U6 stem I annealing (36,48). Subsequent formation of U4/U6 stem II might then lead to displacement of Prp24 and complete U4/U6 assembly (49).…”

Section: Discussionmentioning

confidence: 99%

“…Both Prp24-and Brr2-mediated reactions are also modulated by additional factors. Brr2-mediated U4/U6 disruption is influenced by U4/U6-bound proteins, and U6 conformational switching during U4/U6 unwinding may be aided by emerging U6/U2 interactions on Prp8; Prp24-mediated U4/U6 annealing is enhanced by the LSm proteins (36,(50)(51)(52)(53) and possibly Prp3 (30).…”

Section: Discussionmentioning

confidence: 99%

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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.

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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...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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.

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“…The four RNA recognition motif (RRM) domains of Prp24 wrap around spliceosomal U6 snRNA, making extensive contacts with double and single-stranded regions of the RNA (105). Reorientation of these domains is presumably needed to allow remodeling of U6 base pairs (106). Yeast La protein binds the 3′ ends of pol III transcripts (107, 108) and chaperones misfolded pre-tRNAs (109) and other small nuclear RNAs (110).…”

Section: Introductionmentioning

confidence: 99%

Proteins That Chaperone RNA Regulation

Woodson¹,

Panja²,

Santiago-Frangos

2018

Microbiol Spectr

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RNA-binding proteins chaperone the biological functions of non-coding RNA by reducing RNA misfolding, improving matchmaking between regulatory RNA and targets, and exerting quality control over RNP biogenesis. Recent studies of E. coli CspA, HIV NCp and E. coli Hfq, are beginning to show how RNA-binding proteins remodel RNA structures. These different protein families use common strategies for disrupting or annealing RNA double helices, which can be used to understand the mechanisms by which proteins chaperone RNA-dependent regulation in bacteria.

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“…Instead, the telestem may prove most important at other stages in splicing. It has recently been demonstrated that the telestem assists in U4/U6 annealing by the assembly factor Prp24 (14). Thus, while the U6 ISL is an essential component of spliceosomal catalysis, the U6 telestem likely functions in tri-snRNP assembly.…”

Section: Introductionmentioning

confidence: 99%

A multi-step model for facilitated unwinding of the yeast U4/U6 RNA duplex

et al. 2016

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The small nuclear RNA (snRNA) components of the spliceosome undergo many conformational rearrangements during its assembly, catalytic activation and disassembly. The U4 and U6 snRNAs are incorporated into the spliceosome as a base-paired complex within the U4/U6.U5 small nuclear ribonucleoprotein (tri-snRNP). U4 and U6 are then unwound in order for U6 to pair with U2 to form the spliceosome's active site. After splicing, U2/U6 is unwound and U6 annealed to U4 to reassemble the tri-snRNP. U6 rearrangements are crucial for spliceosome formation but are poorly understood. We have used single-molecule Förster resonance energy transfer and unwinding assays to identify interactions that promote U4/U6 unwinding and have studied their impact in yeast. We find that U4/U6 is efficiently unwound using DNA oligonucleotides by coupling unwinding of U4/U6 stem II with strand invasion of stem I. Unwinding is stimulated by the U6 telestem, which transiently forms in the intact U4/U6 RNA complex. Stabilization of the telestem in vivo results in accumulation of U4/U6 di-snRNP and impairs yeast growth. Our data reveal conserved mechanisms for U4/U6 unwinding and indicate telestem dynamics are critical for tri-snRNP assembly and stability.

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Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly

Cited by 24 publications

References 46 publications

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

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

Proteins That Chaperone RNA Regulation

A multi-step model for facilitated unwinding of the yeast U4/U6 RNA duplex

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