Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Nov 4;47(19):10327-10339.
doi: 10.1093/nar/gkz733.

Functional analysis of Cwc24 ZF-domain in 5' splice site selection

Nan-Ying Wu  1 , Soo-Chen Cheng  1
Affiliations

Functional analysis of Cwc24 ZF-domain in 5' splice site selection

Nan-Ying Wu et al. Nucleic Acids Res. .

Abstract

The essential splicing factor Cwc24 contains a zinc-finger (ZF) domain required for its function in splicing. Cwc24 binds over the 5' splice site after the spliceosome is activated, and its binding prior to Prp2-mediated spliceosome remodeling is important for proper interactions of U5 and U6 with the 5' splice site sequence and selection of the 5' splice site. Here, we show that Cwc24 transiently interacts with the 5' splice site in formation of the functional RNA catalytic core during spliceosome remodeling, and the ZF-motif is required for specific interaction of Cwc24 with the 5' splice site. Deletion of the ZF domain or mutation of the conserved ZF residues greatly weakened the association of Cwc24 with the spliceosome, and lowered the affinity and specificity of its interaction with the 5' splice site, resulting in atypical interactions of U5, U6 and Prp8 with the 5' splice site, and aberrant cleavage at the 5' splice site. Our results reveal a crucial role of the Cwc24 ZF-motif for defining 5' splice site selection in the first splicing step.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Schematic for the spliceosome assembly pathway showing splicing factors recruited to and released from the spliceosome at each stage. Cwc22, Cwc24, Cwc27 and Yju2 are recruited to the spliceosome immediately after or concomitant with binding of NTC. Slu7 and Prp18 are required for the second catalytic step, but can be recruited to the spliceosome at earlier steps. DExD/H-box proteins, Prp5, Sub2, Prp28, Brr2, Prp2 and Prp16, required for transition between specific steps are also shown.
Figure 2.
Figure 2.
The ZF domain is required for the interaction of Cwc24 with the spliceosome, but not with Brr2. (A) Schematic representations of domain structures of Cwc24 and its human (RNF113A) and Drosophila (mdlc) orthologs, and Cwc24 deletion mutants. (B) Splicing reactions were carried out in Spp2-depleted ΔCwc24 extracts supplemented with recombinant V5-tagged Cwc24 proteins at final concentrations of 0.35, 3.5, 35 and 350 nM. The reaction mixtures were precipitated with anti-V5 antibody. The control reaction was precipitated with anti-Ntc20 antibody. ΔCwc24, extracts metabolically depleted of Cwc24; α-20, anti-Ntc20; FL, full-length. (C) Two-hybrid interactions of Cwc24 with segments of Brr2 fused to the DNA-binding domain. AD-Cwc24, Cwc24 fused to the activation domain; Vec, vector; FL, full-length. (D) Two-hybrid interactions of Brr2C with deletion mutants of Cwc24 fused to the activation domain. BD-Brr2C, Brr2C fused to the DNA-binding domain. Vec, vector; FL, full-length. (E) Interactions of Cwc24 deletion mutants with Brr2C were analyzed by immnuoprecipitation with anti-V5 antibody against bacterially expressed V5-tagged Cwc24 and His-SUMO-tagged Brr2C, followed by western blotting probed with anti-His (upper panel) and anti-V5 (lower panel) antibodies. IgG-H, IgG heavy chain; IgG-L, IgG light chain.
Figure 3.
Figure 3.
The ZF domain is required for specific interaction of Cwc24 with the 5′ splice site. (A and B) UV-crosslinking of Cwc24 deletion and C144A mutant proteins to pre-mRNA on the activated spliceosome. Splicing was carried out in Spp2-depleted ΔCwc24 extracts supplemented with recombinant V5-tagged Cwc24 proteins at a final concentration of 35 nM. The reaction mixtures were UV irradiated and then precipitated with anti-V5 antibody with prior denaturation. Reaction mixtures without UV irradiation were also precipitated with or without prior denaturation as controls. The relative amounts of samples loaded onto each lane are 1:10:1000 for RXN:no denat/IP:denat/IP. RXN, reaction mixture; Denat, denaturation. (C) Primer extension analysis of Cwc24 crosslinked sites. Splicing reactions and UV-crosslinking were carried out as in panel (B). RNA was extracted from the reaction mixtures (RNA), or after precipitation with anti-V5 antibody without (RNP) or with prior denaturant treatment (Cwc24) for primer extension using oligo Pre-V as a primer.
Figure 4.
Figure 4.
The Cwc24 C144A mutation altered U5-5′SS interactions. (A) Splicing was carried out in wild-type or ΔCwc24 extracts supplemented with or without recombinant V5-tagged wild-type or C144A Cwc24 protein at a concentration of 35 or 3,500 nM, and the reaction mixtures were precipitated with anti-Ntc20 antibody. Following UV-irradiation and deproteinization, U6- and U5-crosslinked products were affinity-selected with biotinylated oligos U6-Abio and U5-Cbio, respectively, and analyzed by PAGE. (B) Individual U5-crosslinked products from lanes 8, 9 and 11 of (A) were excised from the gel and RNA extracted for primer extension analysis using oligo Pre-III as a primer. 1, 3, 4 indicate Y1, Y3 and Y4, respectively.
Figure 5.
Figure 5.
Cwc24 ZF mutants exhibit dominant-negative properties. (A) UV-crosslinking of Cwc24. Splicing was carried out in ΔCwc24 extracts, and the reaction mixtures were depleted of ATP before addition of recombinant V5-tagged wild-type or ΔZF Cwc24 protein at a final concentration of 3.5 μM. The reaction mixtures were UV irradiated and then precipitated with anti-V5 antibody with prior denaturation. Reaction mixtures without UV irradiation were also precipitated with or without prior denaturation as controls. The relative amounts of samples loaded onto each lane were 1:10:1000 for RXN:no denat/IP:denat/IP. RXN, reaction mixture; PAS, protein A-Sepharose; Denat, denaturation; FL, full-length. (B) Splicing reactions were carried out in the presence of recombinant Cwc24 wild-type, ΔZF or C144A mutant protein at concentrations ranging from 0.035 to 3500 nM. (C) Splicing was carried out in the presence of recombinant V5-tagged wild-type or mutant Cwc24 proteins at a concentration of 3.5 μM, and the reaction mixtures were precipitated with anti-Ntc20 or anti-V5 antibody; RXN, reaction.
Figure 6.
Figure 6.
The dominant-negative Cwc24 ZF mutant exhibits aberrant cleavage at the 5′ splice site. Splicing was carried out with wild-type or G5A actin pre-mRNA in the absence or presence of 35 to 3500 nM wild-type or C144A V5-tagged Cwc24, and the reaction mixtures were precipitated with anti-Ntc20 antibody.
Figure 7.
Figure 7.
Cwc24 modulates the interaction of Prp8 with the 5′ splice site. Primer extension analyses to map Prp8-crosslinked sites at the 5′ splice site using oligo Pre-II as a primer. (A) Splicing was carried out in Cwc24-depleted or Spp2-depleted extracts. The reaction mixtures were UV-irradiated and precipitated with or without anti-Prp8 antibody-conjugated PAS with or without prior denaturant treatment. RNA was extracted for primer extension analysis; PAS, protein A-Sepharose. (B) Splicing was carried out in Prp2-depleted ΔCwc24 extracts supplemented with recombinant V5-tagged wild-type or C144A Cwc24 protein at a final concentration of 35 nM. The reaction mixtures were UV-irradiated (lanes 1 and 4), and Cwc24-associated (lane 2) and Cwc24-lacking (lane 3) spliceosomes were separated by precipitation with anti-V5 antibody. Following denaturant treatment, samples were precipitated with anti-Prp8 antibody and RNA was extracted for primer extension analysis; P, pellet; S, supernatant. (C) Splicing was carried out in NTC-depleted (lane 1), Spp2-depleted (lane 2), Cwc24-depleted (lane 3) or Cwc25-depleted (lane 4) extracts. The reaction mixtures were UV-irradiated, precipitated with anti-Prp8 antibody following denaturant treatment, and then RNA was extracted for primer extension analysis.
Figure 8.
Figure 8.
A model for how Cwc24 may modulate formation of the RNA catalytic core. During spliceosome activation, NTC is recruited to the spliceosome upon the release of U4. In the presence of Cwc24, which may bind to the spliceosome immediately upon binding of NTC (32), U5 and U6 interact with the 5′ splice site at correct positions to form an active catalytic core, with U5 interacting with the -1 position and U6 forming base pairs between residues ACAGA and UGU of the intron. The ZF domain of Cwc24 sequesters Prp8 from interacting with the GU residues of the 5′ splice site. The mode of RNA interactions is retained after Prp2-mediated spliceosome remodeling, which results in the release of Cwc24 to allow Prp8 to interact with the +1 and +2 positions of the 5′ splice site. When Cwc24 is absent during spliceosome activation, Prp8 interacts with the 5′ splice site in a dynamic manner over a wider region and U5-5′SS and U6-5′SS interactions are misarranged.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Brow D.A. Allosteric cascade of spliceosome activation. Annu. Rev. Genet. 2002; 36:333–360. - PubMed
    1. Wahl M.C., Will C.L., Lührmann R.. The spliceosome: design principles of a dynamic RNP machine. Cell. 2009; 136:701–718. - PubMed
    1. Will C.L., Lührmann R.. Atkins JF, Gesteland RF, Cech TR. Cold Spring Harb. Perspect. Biol. 2011; 3:NY: Cold Spring Harbor Laboratory Press; a003707. - PubMed
    1. Fica S.M., Tuttle N., Novak T., Li N.-S., Lu J., Koodathingal P., Dai Q., Staley J.P., Piccirilli J.A.. RNA catalyzes nuclear pre-mRNA splicing. Nature. 2013; 503:229–234. - PMC - PubMed
    1. Cordin O., Beggs J.D.. RNA helicases in splicing. RNA Biol. 2013; 10:83–95. - PMC - PubMed

Publication types

MeSH terms