Mutations in pre-mRNA processing factors (PRPFs) cause autosomal-dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause non-syndromic retinal disease. Here, we generate transcriptome profiles from RP11 (PRPF31-mutated) patient-derived retinal organoids and retinal pigment epithelium (RPE), as well as Prpf31+/− mouse tissues, which revealed that disrupted alternative splicing occurred for specific splicing programmes. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and Prpf31+/− mouse retinae and RPE. Mis-splicing of genes implicated in ciliogenesis and cellular adhesion was associated with severe RPE defects that include disrupted apical – basal polarity, reduced trans-epithelial resistance and phagocytic capacity, and decreased cilia length and incidence. Disrupted cilia morphology also occurred in patient-derived photoreceptors, associated with progressive degeneration and cellular stress. In situ gene editing of a pathogenic mutation rescued protein expression and key cellular phenotypes in RPE and photoreceptors, providing proof of concept for future therapeutic strategies.

Assembly of a spliceosome, catalyzing precursor-messenger RNA splicing, involves multiple RNA-protein remodeling steps, driven by eight conserved DEXD/H-box RNA helicases. The 250-kDa Brr2 enzyme, which is essential for U4/U6 di-small nuclear ribonucleoprotein disruption during spliceosome catalytic activation and for spliceosome disassembly, is the only member of this group that is permanently associated with the spliceosome, thus requiring its faithful regulation. At the same time, Brr2 represents a unique subclass of superfamily 2 nucleic acid helicases, containing tandem helicase cassettes. Presently, the mechanistic and regulatory consequences of this unconventional architecture are unknown. Here we show that in human Brr2, two ring-like helicase cassettes intimately interact and functionally cooperate and how retinitis pigmentosa-linked Brr2 mutations interfere with the enzyme's function. Only the N-terminal cassette harbors ATPase and helicase activities in isolation. Comparison with other helicases and mutational analyses show how it threads single-stranded RNA, and structural features suggest how it can load onto an internal region of U4/U6 di-snRNA. Although the C-terminal cassette does not seem to engage RNA in the same fashion, it binds ATP and strongly stimulates the N-terminal helicase. Mutations at the cassette interface, in an intercassette linker or in the C-terminal ATP pocket, affect this cross-talk in diverse ways. Together, our results reveal the structural and functional interplay between two helicase cassettes in a tandem superfamily 2 enzyme and point to several sites through which Brr2 activity may be regulated.pre-mRNA splicing | RNA helicase Brr2 | X-ray crystallography N ucleotide triphosphate-dependent nucleic acid unwindases ("helicases") serve as motors and regulators of many biological macromolecular machines. Assembly of a spliceosome, catalyzing precursor-messenger RNA splicing, is a paradigmatic case that involves multiple RNA-protein remodeling steps, driven by eight conserved RNA helicases of the DEXD/H-box family (1). None of the spliceosome's small nuclear ribonucleoprotein (snRNP) subunits (U1, U2, U4, U5, and U6 in the major spliceosome) or its plethora of non-snRNP factors bear a preformed active center for splicing catalysis. Instead, profound compositional and conformational changes are required to convert an initial, inactive assembly to a catalytically competent spliceosome (2).Catalytic activation involves the unwinding of the U4 and U6 snRNAs, which are extensively base-paired via two regions (stems 1 and 2) when delivered to the spliceosome in the framework of the U4/U6-U5 tri-snRNP. As the U5 snRNP protein, Brr2, unwinds U4/U6 duplexes in vitro (3, 4) and Brr2 mutations interfere with catalytic activation (5-7), the enzyme is thought to elicit these rearrangements. Brr2 already encounters its U4/U6 substrate in the U4/U6-U5 tri-snRNP, but U4/U6 dissociation must be delayed until splice sites have been reliably located during spliceosome assembly. Furthermore, unl...

The Ski2-like RNA helicase Brr2 is a core component of the spliceosome that must be tightly regulated to ensure correct timing of spliceosome activation. Little is known about mechanisms of regulation of Ski2-like helicases by protein cofactors. Here we show by crystal structure and biochemical analyses that the Prp8 protein, a major regulator of the spliceosome, can insert its C-terminal tail into Brr2's RNA-binding tunnel, thereby intermittently blocking Brr2's RNA-binding, adenosine triphosphatase, and U4/U6 unwinding activities. Inefficient Brr2 repression is the only recognizable phenotype associated with certain retinitis pigmentosa-linked Prp8 mutations that map to its C-terminal tail. Our data show how a Ski2-like RNA helicase can be reversibly inhibited by a protein cofactor that directly competes with RNA substrate binding.

The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 disnRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation.[Keywords: pre-mRNA splicing; RNA helicase structure and function; remodeling of RNA-protein complexes; spliceosome catalytic activation; X-ray crystallography] Supplemental material is available for this article.Received September 14, 2015; revised version accepted November 13, 2015.Splicing entails the removal of noncoding sequences (introns) from primary transcripts and the concomitant ligation of neighboring coding regions (exons). It is mediated by a highly dynamic, multimegadalton RNA protein (RNP) molecular machine, the spliceosome, which consists of five small nuclear RNPs (snRNPs; U1, U2, U4, U5, and U6 in the case of the major spliceosome) and numerous non-snRNPs (Wahl et al. 2009). For each round of splicing, a spliceosome is assembled de novo on a substrate by the stepwise recruitment of snRNPs and nonsnRNPs. After assembly of a precatalytic complex, the spliceosome is catalytically activated and carries out the two consecutive steps of a splicing reaction before it is disassembled and its subunits are recycled. Each assembly, activation, catalysis, and disassembly step involves profound rearrangements of the spliceosomal RNP interaction networks, mediated predominantly by eight conserved superfamily 2 (SF2) NTPases/RNA helicases (Staley and Guthrie 1998). The most extensive rearrangements occur during spliceosome activation. In the precatalytic spliceosome, U4 and U6 snRNPs form a disnRNP by base-pairing of their snRNAs and are associated with U5 snRNP via protein-protein interactions. During spliceosome activation, the U5 snRNP-specific Brr2 helicase unwinds the U4/U6 ...