Mechanism of 5' splice site transfer for human spliceosome activation

Abstract

The prespliceosome, comprising U1 and U2 small nuclear ribonucleoproteins (snRNPs) bound to the precursor messenger RNA 5' splice site (5'SS) and branch point sequence, associates with the U4/U6.U5 tri-snRNP to form the fully assembled precatalytic pre-B spliceosome. Here, we report cryo-electron microscopy structures of the human pre-B complex captured before U1 snRNP dissociation at 3.3-angstrom core resolution and the human tri-snRNP at 2.9-angstrom resolution. U1 snRNP inserts the 5'SS-U1 snRNA helix between the two RecA domains of the Prp28 DEAD-box helicase. Adenosine 5'-triphosphate-dependent closure of the Prp28 RecA domains releases the 5'SS to pair with the nearby U6 ACAGAGA-box sequence presented as a mobile loop. The structures suggest that formation of the 5'SS-ACAGAGA helix triggers remodeling of an intricate protein-RNA network to induce Brr2 helicase relocation to its loading sequence in U4 snRNA, enabling Brr2 to unwind the U4/U6 snRNA duplex to allow U6 snRNA to form the catalytic center of the spliceosome.

Fig. 1. Structures of human tri-snRNP and pre-B complex.

(A) Overview of human U4/U6.U5 tri-snRNP. (B and C) Overview of the human U1.U2.U4/U6.U5 pre-B complex in two orientations. The black dashed line represents the theoretical path of the pre-mRNA intron.

Fig. 2. Structural features of the human tri-snRNP.

(A) Organization of U4 snRNA in the tri-snRNP prior to Brr2 relocation. SL, stem loop. The mobile ACAGAGA loop is depicted as a red dashed line. (B) Close-up view of U4/U6 stem I capped by the quasi-pseudoknot and its stabilization by RBM42 RRM. (C) and (D) U4 region in two orientations. Note how Snu66 and SNRNP-27K wrap around the U4 Sm ring, Prp8 Endonuclease and RNaseH domains, U4/U6 stem III and the quasi-pseudoknot thereby solidifying the organization of the U4 region prior to Brr2 relocation. (E) Complex interaction network of Brr2 N-terminal domain with tri-snRNP components. Brr2-C, C-terminal helicase cassette of Brr2. Brr2-N, N-terminal helicase cassette of Brr2. Prp8-L, large domain of Prp8. (F) Another view of the complex interaction network of the Brr2 N-terminal domain with tri-snRNP components. Prp8-RT, Prp8 reverse transcriptase domain. Note the extended N-terminal domain of Prp6. On this panel, the Prp4 kinase (active site is represented as an orange circle) as it interacts in pre-B and its phosphorylation targets on Prp6 (magenta spheres) are represented.

Fig. 3. Interaction of Prp28 with the tri-snRNP.

(A) Interaction of the N-terminus of Prp28 with the tri-snRNP. Brr2-C, C-terminal helicase cassette of Brr2. Prp8-N, N-terminal domain of Prp8. (B) Same view but of the 5′ exon and B-complex proteins in human B complex (PDB 6AHD). (C) Sequence alignment of Prp28 N-termini from different species.

Fig. 4. Binding of U1 snRNP to tri-snRNP in pre-B complex.

(A) Close-up view of the U1 / tri-snRNP interface in pre-B complex. The mobile ACAGAGA loop is depicted as a red dashed line. The disordered U5 snRNA loop 1 is depicted as a blue dashed line. The 5′ splice site (5′SS) is in striking proximity to the ACAGAGA sequence, looped out between the quasi-pseudoknot and U4/U6 stem III, and U5 snRNA loop 1. (B) A model of Prp28 with the 5′ end of U1 snRNA after ATP-dependent closure of the two RecA domains and the 5′SS release. The RecA1 domain of the closed form of the Vasa DEAD-box helicase was overlaid on the RecA1 domain of Prp28, showing the position of the 5′ end of U1 snRNA. (C) Superposition of the closed form of the Vasa DEAD-box helicase on Prp28 RecA1, showing the position of the ssRNA as observed in the original crystal structure (26). Note that the ssRNA and the 5′ end of U1 snRNA occupy a similar position suggesting that Prp28 disrupts the U1/5′SS duplex by binding the U1 snRNA, thereby freeing the pre-mRNA 5′SS to pair with the free ACAGAGA sequence.

Fig. 5. Schematic representation of pre-B complex activation.

(A) Association of the prespliceosome with the tri-snRNP. (B) Docking of U1 snRNP inserts the 5′SS-U1 snRNA duplex between the two RecA domains of Prp28. (C) Transfer of the 5′SS to the U6 snRNA ACAGAGA-loop destabilizes the binding of U1 snRNP and Prp28. (D) Formation of the 5′SS-ACAGAGA helix and dissociation of Prp28 induce movement of the U4 core domain and relocation of the Brr2 helicase. (E) Binding of Brr2 to the single stranded region of U4 snRNA made accessible by the 5′SS induced reorganization of the U4-U6 snRNA interaction.