Research Article

Structure of the activated human minor spliceosome

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Science  19 Mar 2021:
Vol. 371, Issue 6535, eabg0879
DOI: 10.1126/science.abg0879

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Atomic structure of the minor spliceosome

About 1% of the human genome contains the so-called U12-type introns, which are spliced by the minor spliceosome. Compared with the major spliceosome, the composition, assembly, functional states, activation, regulation, and structure of the minor spliceosome have been enigmatic. Bai et al. assembled the activated human minor spliceosome in an in vitro U12-type intron-splicing assay and determined the structure by cryo–electron microscopy at 2.9-angstrom resolution. They found a number of exciting and unanticipated features, including five new proteins that play an important role in the activated minor spliceosome.

Science, this issue p. eabg0879

Structured Abstract

INTRODUCTION

Precursor messenger RNA (pre-mRNA) splicing, which involves the removal of noncoding introns and the ligation of the coding exons, is achieved by the spliceosome. An intron is defined by a 5′ splice site (5′SS), a branch point sequence (BPS), and a 3′ splice site (3′SS). Most introns belong to the U2 type and are removed by the major spliceosome that contains the U2 small nuclear RNA (snRNA). A very small percentage of introns are the U12 type, which is characterized by a distinct 5′SS and BPS. The U12-type genes, present in all major eukaryotic taxa, play an essential role in development. The U12-type intron is removed by the minor spliceosome, which contains five snRNAs: U11, U12, U4atac, U5, and U6atac. Of these snRNAs, only U5 is shared between the major and minor spliceosomes.

Because of its scarcity in cells, the minor spliceosome has represented a challenge for biochemical studies. Before now, there was no structural information or published protocol on the purification of the minor spliceosome. It was unclear how many U12-specific protein components are present in each major functional state of the minor spliceosome and how they may facilitate the splicing reaction. We did not even know whether rules derived from the major spliceosome could apply to the minor spliceosome.

RATIONALE

To address these questions, we need to both develop a protocol for the assembly and purification of the minor spliceosome and determine the structure of the minor spliceosome at resolutions that give sufficiently detailed structural features. Structural comparison between the major and minor spliceosomes may reveal valuable information on protein components specific to either spliceosome, U12-type intron recognition, snRNA conformation, active site configuration, and regulatory mechanisms.

RESULTS

We replaced the U2-type 5′SS, BPS, and 3′SS of the intron in the MINX pre-mRNA with those of the U12-type, generating a MINX-U12 pre-mRNA. In an in vitro splicing assay, MINX-U12 undergoes splicing to produce ligated exons under conditions in which the major spliceosomes are inactivated in the nuclear extract. These results demonstrate the presence of active minor spliceosome in the extract. We then truncated MINX-U12 such that the binding site for the ATPase/helicase PRP2 is absent. This strategy, which was designed to enrich the activated minor spliceosome (Bact complex) by preventing its remodeling by PRP2, proved to be successful. We purified the human minor Bact complex and determined its cryo–electron microscopy (cryo-EM) structure at an average resolution of 2.9 Å.

Although the overall organization of the RNA elements in the human minor Bact complex closely resembles that in the major Bact complex, there are notable local differences. Compared with U6 snRNA in the major spliceosome, U6atac snRNA lacks the 5′ stem loop, and the U12/U6atac duplex lacks helix II. Notably, the 3′-end sequences of U6atac snRNA form a characteristic 3′ stem loop that is placed in approximately the same location as that of the U2/U6 helix II in the major Bact complex. The distinct 5′SS and BPS of the U12-type intron are recognized through extensive base-pairing interactions by U6atac and U12 snRNA, respectively. Two catalytic metals, M1 and M2, are already loaded in the splicing active site center and poised for catalysis of the branching reaction.

The EM maps allow for the identification of five previously unidentified proteins—SCNM1, RBM48, ARMC7, CRIPT, and PPIL2—that appear to play key roles in the minor spliceosome. SCNM1 mimics the SF3a complex of the major spliceosome. The N-terminal domain of SCNM1 shares sequence homology with SF3a66 of the SF3a complex. Similarly to SF3a66, the N-terminal domain of SCNM1 binds the BPS/U12 duplex and the proteins SF3b155, SF3b145, and CDC5L, whereas the N terminus inserts into the active site to interact with 5′SS, U6atac snRNA, and the splicing factor RNF113A. The C-terminal domain of SCNM1 functionally mimics SF3a60 (another component of the SF3a complex). The RBM48-ARMC7 complex binds the γ-monomethyl phosphate cap of the guanine nucleotide at the 5′ end of U6atac snRNA through extensive interactions. The splicing factor CRIPT binds RNF113A and stabilizes U12 small nuclear ribonucleoprotein (snRNP) through interactions with SF3b14b, SF3b145, and SF3b155. The U-box protein PPIL2 in an extended conformation interacts with a number of proteins and RNA, stabilizing the overall conformation of U5 snRNP. The N terminus of PPIL2 specifically recognizes loop I of U5 snRNA. Additionally, PRP2 and its coactivator SPP2 are bound to the minor Bact complex, which suggests similar roles for PRP2 and SPP2 in the minor spliceosome as those observed in the major spliceosome.

CONCLUSION

The cryo-EM structure of the human minor Bact complex reveals a number of previously unknown features, including the identification of minor spliceosome–specific proteins. This structure serves as a framework for the mechanistic understanding of the functions of the minor spliceosome.

Structure of the activated human minor spliceosome (Bact complex).

(Left) The minor Bact complex comprises U12 snRNP, U5 snRNP, U6atac snRNA, the truncated MINX-U12 pre-mRNA, nineteen complex (NTC), NTC-related (NTR), the retention and splicing (RES) complex, two prolyl peptidyl isomerase (PPIase)–like proteins (PPIs), and nine splicing factors. (Right) Based on EM maps, the minor spliceosome contains five newly identified proteins—SCNM1, RBM48, ARMC7, CRIPT, and PPIL2—which together with snRNAs are highlighted in the foreground. All other components are in the background.

Abstract

The minor spliceosome mediates splicing of the rare but essential U12-type precursor messenger RNA. Here, we report the atomic features of the activated human minor spliceosome determined by cryo–electron microscopy at 2.9-angstrom resolution. The 5′ splice site and branch point sequence of the U12-type intron are recognized by the U6atac and U12 small nuclear RNAs (snRNAs), respectively. Five newly identified proteins stabilize the conformation of the catalytic center: The zinc finger protein SCNM1 functionally mimics the SF3a complex of the major spliceosome, the RBM48-ARMC7 complex binds the γ-monomethyl phosphate cap at the 5′ end of U6atac snRNA, the U-box protein PPIL2 coordinates loop I of U5 snRNA and stabilizes U5 small nuclear ribonucleoprotein (snRNP), and CRIPT stabilizes U12 snRNP. Our study provides a framework for the mechanistic understanding of the function of the human minor spliceosome.

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