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 ...

Structural rearrangement of the activated spliceosome (B act ) to yield a catalytically active complex (B*) is mediated by the DEAH-box NTPase Prp2 in cooperation with the G-patch protein Spp2. However, how the energy of ATP hydrolysis by Prp2 is coupled to mechanical work and what role Spp2 plays in this process are unclear. Using a purified splicing system, we demonstrate that Spp2 is not required to recruit Prp2 to its bona fide binding site in the B act spliceosome. In the absence of Spp2, the B act spliceosome efficiently triggers Prp2's NTPase activity, but NTP hydrolysis is not coupled to ribonucleoprotein (RNP) rearrangements leading to catalytic activation of the spliceosome. Transformation of the B act to the B* spliceosome occurs only when Spp2 is present and is accompanied by dissociation of Prp2 and a reduction in its NTPase activity. In the absence of spliceosomes, Spp2 enhances Prp2's RNA-dependent ATPase activity without affecting its RNA affinity. Our data suggest that Spp2 plays a major role in coupling Prp2's ATPase activity to remodeling of the spliceosome into a catalytically active machine.[Keywords: spliceosome activation; DEAH-box helicase; Prp2; G-patch protein; Spp2; ATP hydrolysis] Supplemental material is available for this article. Pre-mRNA splicing proceeds by way of two phosphoester transfer reactions and is catalyzed by the spliceosome, which consists of the U1, U2, U4/U6, and U5 small nuclear ribonucleoproteins (snRNPs) and numerous nonsnRNP proteins (Wahl et al. 2009). Spliceosome assembly occurs de novo on each pre-mRNA and follows an intricate pathway involving major structural rearrangements during each round of splicing. The various remodeling steps are driven in yeast by eight conserved DExD/H-box ATPases/ RNA helicases. An interesting feature of the spliceosome is that it initially assembles into a multimegadalton ensemble-termed complex B-that contains all of the snRNPs but does not yet have an active site. Activation of the spliceosome is then initiated by the combined action of the Prp28 and Brr2 RNA helicases, yielding the B act complex. In this process, U1 and U4 snRNPs are displaced from the spliceosome, and new base-pair interactions between the U6 and U2 snRNAs and between U6 and the 59 splice site (59SS) are formed. The resulting RNA structure plays a central role in catalyzing both steps of pre-mRNA splicing (Staley and Guthrie 1998;Fica et al. 2013). During activation, 20 new proteins, including those of the NTC (nineteen complex), are stably integrated into the B act complex and stabilize the newly formed RNA-RNA interaction network (Chan et al. 2003;Chan and Cheng 2005;Fabrizio et al. 2009). The final catalytic activation of the spliceosome requires an additional ATP-dependent remodeling step, yielding complex B*. This step is catalyzed by the DEAH-box ATPase Prp2 (Kim and Lin 1996).Prp2 is structurally related to three other spliceosomal DEAH-box ATPases: Prp16, Prp22, and Prp43, which are involved, respectively, in the second catalytic step, the

Brr2 is a unique DExD/H box protein required for catalytic activation and disassembly of the spliceosome. It contains two tandem helicase cassettes that both comprise dual RecA-like domains and a noncanonical Sec63 unit. The latter may bestow the enzyme with unique properties. We have determined crystal structures of the C-terminal Sec63 unit of yeast Brr2, revealing three domains, two of which resemble functional modules of a DNA helicase, Hel308, despite lacking significant sequence similarity. This structural similarity together with sequence conservation between the enzymes throughout the RecA-like domains and a winged helix domain allowed us to devise a structural model of the N-terminal active cassette of Brr2. We consolidated the model by rational mutagenesis combined with splicing and U4/U6 di-snRNA unwinding assays, highlighting how the RecA-like domains and the Sec63 unit form a functional entity that appears suitable for unidirectional and processive RNA duplex unwinding during spliceosome activation and disassembly.

The spliceosomal RNA helicase Brr2 catalyzes unwinding of the U4/U6 snRNA duplex, an essential step for spliceosome catalytic activation. Brr2 is regulated in part by the spliceosomal Prp8 protein by an unknown mechanism. We demonstrate that the RNase H (RH) domain of yeast Prp8 binds U4/U6 small nuclear RNA (snRNA) with the single-stranded regions of U4 and U6 preceding U4/U6 stem I, contributing to its binding. Via cross-linking coupled with mass spectrometry, we identify RH domain residues that contact the U4/U6 snRNA. We further demonstrate that the same single-stranded region of U4 preceding U4/U6 stem I is recognized by Brr2, indicating that it translocates along U4 and first unwinds stem I of the U4/U6 duplex. Finally, we show that the RH domain of Prp8 interferes with U4/U6 unwinding by blocking Brr2's interaction with the U4 snRNA. Our data reveal a novel mechanism whereby Prp8 negatively regulates Brr2 and potentially prevents premature U4/U6 unwinding during splicing. They also support the idea that the RH domain acts as a platform for the exchange of U6 snRNA for U1 at the 59 splice site. Our results provide insights into the mechanism whereby Brr2 unwinds U4/U6 and show how this activity is potentially regulated prior to spliceosome activation.[Keywords: pre-mRNA splicing; RNA helicase; RNA-protein complex; RNA-protein cross-linking; spliceosome catalytic activation] Supplemental material is available for this article. Received July 11, 2012; revised version accepted September 18, 2012. Pre-mRNA splicing is catalyzed by a multisubunit RNAprotein enzyme, the spliceosome, which carries out two successive trans-esterification reactions that lead to removal of an intron and the ligation of its flanking exons. Spliceosomes are formed via the stepwise recruitment of small nuclear ribonucleoprotein particles (snRNPs) and numerous non-snRNP proteins to the pre-mRNA substrate (for review, see Wahl et al. 2009). Initially, U1 and U2 snRNPs bind the 59 splice site (59 SS) and the branch site (BS) of the pre-mRNA's intron, respectively. This is followed by the recruitment of the U4/U6.U5 tri-snRNP to the spliceosome, yielding the B complex, which does not yet have an active center. The U4 and U6 snRNAs are extensively base-paired in the tri-snRNP, thereby keeping U6 small nuclear RNA (snRNA) catalytically inert (Staley and Guthrie 1998). For catalytic activation of the spliceosome, U4 snRNA must be displaced from U6 snRNA, which allows the formation of new U2/U6 base-pairing interactions and a catalytically important U6 internal stem-loop (ISL) ). Concomitant with or prior to this, the base-pairing interaction between the U1 snRNA and the 59 SS must be disrupted to allow the highly conserved ACAGAGA sequence of U6 snRNA to base-pair with the 59 end of the intron. This newly formed U2-U6-pre-mRNA RNA interaction network is thought to comprise the heart of the catalytic center of the spliceosome (Madhani and Guthrie 1992;Villa et al. 2002).Brr2 catalyzes the dissociation of the U4/U6 duplex and thus plays a ke...

In our study of the spliceosomal Brr2 helicase, we incorrectly labeled a fragment of the protein Prp8 that we used in activity assays of Brr2 and Brr2 mutants. Throughout the main text and Supplemental Information, we inadvertently referred to the fragment as ''Prp8 (1796-2092)''; however, the correct fragment we used is ''Prp8(1806-2413)''. We apologize for any inconvenience this may have caused.