Functions and regulation of the Brr2 RNA helicase during splicing

Abstract

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 down-regulate Brr2 activity. Biochemical studies revealed an intricate cross-talk among the various cis- and trans-regulatory 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.

Keywords: Pre-mRNA splicing; RNA helicase structure,; function and regulation; regulation of pre-mRNA splicing; remodeling of RNA-protein complexes; spliceosome catalytic activation.

Figure 1.

Top, pre-mRNA splicing by the spliceosome, showing characterized spliceosomal assembly, activation, catalysis and disassembly intermediates (complexes E, A, Pre-B, B, B^act, B*, C, C*, PSC [post-splicing complex] and ILS [intron-lariat spliceosome]). For clarity, the cycle only depicts the compositions with respect to the snRNPs, the many non-snRNP proteins that are also participating have been omitted. Step 1, step 2, first/second transesterification reaction of splicing. Spliceosomal RNA helicases are indicated at the steps during which they are required (blue). Traffic lights refer to the observed or assumed (with question mark) states of Brr2 at the respective stages and during the various transitions (red, fully inhibited; yellow, partially inhibited, green, activated). Bottom, schemes illustrating combinatorial regulation of Brr2 via its NTR (magenta) and via the Prp8 Jab1 domain (gold).

Figure 2.

Domain organization and structure of Brr2 in complex with the Prp8 Jab1 domain. (A) Domain organization of the NTR and NC; the CC has the same domain organization as the NC and is not depicted in detail. (B) Diametric view of a full-length yeast Brr2-Jab1 complex (PDB ID 5M52). The NTR and the Jab1 domain are highlighted by semi-transparent surfaces. NTR, magenta; RecA1, light gray; RecA2, dark gray; WH, black; HB, blue; HLH, red; IG, green; CC, beige; Jab1, gold. (C) Accommodation of the U4/U6 di-snRNA substrate by Brr2 as seen in a yeast tri-snRNP structure (PDB ID 5GAO). Color coding as in (B). The rotation symbol indicates the orientation relative to (B), left panel. (D) Interactions involving the E890 residue of human Brr2 (equivalent to E909 in yeast Brr2) in a crystal structure with ADP bound at the NC (PDB ID 4F93). Dashed lines, hydrogen bonds or salt bridges. The rotation symbol indicates the orientation relative to (C).

Figure 3.

Comparison of the N-terminal extensions of various SF2 helicases. Structures were spatially aligned with respect to their RecA1 domains. (A) Truncated yeast Brr2-Jab1 (PDB ID 5M5P). (B) Yeast Prp5 (PDB ID 4LYJ). (C) Human DDX19 (PDB ID 3EWS). (D) Yeast Prp43 (PDB ID 2XAU). (E) Yeast Mtr4 (PDB ID 4QU4). (F) N. crassa FRH bound to ADP (PDB ID 5E02). The color code for the various domains and regions is indicated on the right. Rotation symbols indicate views relative to panel (A).

Figure 4.

State of Brr2 in isolation and in tri-snRNPs. Diametric views of (A) an isolated yeast Brr2-Jab1 complex (PDB ID 5DCA), (B) human U4/U6•U5 tri-snRNP (PDB ID 3JCR) and (C) yeast U4/U6•U5 tri-snRNP (PDB ID 5GAN). Brr2, U4 snRNA, U6 snRNA and proteins interacting with the Brr2 NTR or the CC are highlighted by colors. NTR, magenta; NC, dark gray; CC, beige; Prp8 RH domain, light orange; Jab1, gold; Snu114, dark red; Sad1, cyan; Prp6, dark green; Snu66, light green; Prp3, dark blue; U4 snRNA, brown; U6 snRNA, orange. Green asterisks in (B), right panel, location of the Brr2 active site; orange asterisks in (B), right panel, approximate location of the U4 central domain, where Brr2 initially binds the U4/U6 duplex. Views with Brr2 in the same orientation as in (A).

Figure 5.

State of Brr2 in different spliceosomal contexts and interaction with other spliceosomal helicases. Views of (A) a yeast spliceosomal B^act complex (PDB ID 5GM6) and (B) a yeast spliceosomal C complex (PDB ID 5LJ5). Brr2 and proteins interacting with the Brr2 NTR, and the Prp2 and Prp16 helicases are highlighted by colors. NTR, magenta; NC, dark gray; CC, beige; Jab1, gold; SF3B1, dark teal; Prp2, red; Prp16, dark red. Views with Brr2 in the same orientation as in Figure 4A. Figure 5A, left, is turned 30° around the vertical axis compared to Figure 4A.

Figure 6.

Model for putative Brr2-mediated enhancement of splicing fidelity and regulation of alternative splicing. Depending on the state of the NTR and the Sad1 protein in competing alternative splicing scenarios (left and right branches), Brr2 may be more or less prone to disrupt the tri-snRNP in a non-productive fashion, thus differentially channeling the different substrates along the splicing or discard pathways. Similarly, depending on the level of Brr2 inhibition in competing alternative splicing scenarios, the helicase may elicit spliceosome activation slowly or quickly, kinetically controlling the levels of protein isoforms produced.