Arresting Spliceosome Intermediates at Various Stages of the Splicing Pathway.

The spliceosome is a dynamic ribonucleoprotein particle and is assembled via sequential binding of five snRNAs and numerous protein factors. To understand the molecular mechanism of the splicing reaction, it is necessary to dissect the spliceosome pathway and isolate spliceosome intermediates in various stages of the pathway for biochemical and structural analysis. Here, we describe protocols for preparing intron-containing transcripts, cell-free splicing extracts, and in vitro splicing reactions, as well as procedures to arrest the spliceosome at different stages of the pathway for characterization of specific splicing complexes from the budding yeast Saccharomyces cerevisiae. Methods for arresting spliceosomes at specific stages include depletion with antibodies against factors required for specific steps of the pathway, use of extracts prepared from temperature-sensitive mutants, use of dominant negative mutants of DExD/H-box proteins, and use of mutant substrates.

A missense variant in the nuclear localization signal of DKC1 causes Hoyeraal-Hreidarsson syndrome.

Hoyeraal-Hreidarsson syndrome (HHS) is the most severe form of dyskeratosis congenita (DC) and is caused by mutations in genes involved in telomere maintenance. Here, we identified male siblings from a family with HHS carrying a hemizygous mutation (c.1345C > G, p.R449G), located in the C-terminal nuclear localization signal (NLS) of the DKC1 gene. These patients exhibit progressive cerebellar hypoplasia, recurrent infections, pancytopenia due to bone marrow failure, and short leukocyte telomere lengths. Single-cell RNA sequencing analysis suggested defects in the NLRP3 inflammasome in monocytes and the activation and maturation of NK cells and B cells. In experiments using induced pluripotent stem cells (iPSCs) from patients, DKC1_R449G iPSCs had short telomere lengths due to reduced levels of human telomerase RNA (hTR) and increased cytosolic proportions of DKC1. Treatment with dihydroquinolizinone RG7834 and 3'deoxyanosine cordycepin rescued telomere length in patient-derived iPSCs. Together, our findings not only provide new insights into immunodeficiency in DC patients but also provide treatment options for telomerase insufficiency disorders.

In vitro genome editing rescues parkinsonism phenotypes in induced pluripotent stem cells-derived dopaminergic neurons carrying LRRK2 p.G2019S mutation.

BACKGROUND: The c.G6055A (p.G2019S) mutation in leucine-rich repeat kinase 2 (LRRK2) is the most prevalent genetic cause of Parkinson's disease (PD). CRISPR/Cas9-mediated genome editing by homology-directed repair (HDR) has been applied to correct the mutation but may create small insertions and deletions (indels) due to double-strand DNA breaks. Adenine base editors (ABEs) could convert targeted A·T to G·C in genomic DNA without double-strand breaks. However, the correction efficiency of ABE in LRRK2 c.G6055A (p.G2019S) mutation remains unknown yet. This study aimed to compare the mutation correction efficiencies and off-target effects between HDR and ABEs in induced pluripotent stem cells (iPSCs) carrying LRRK2 c.G6055A (p.G2019S) mutation. METHODS: A set of mutation-corrected isogenic lines by editing the LRRK2 c.G6055A (p.G2019S) mutation in a PD patient-derived iPSC line using HDR or ABE were established. The mutation correction efficacies, off-target effects, and indels between HDR and ABE were compared. Comparative transcriptomic and proteomic analyses between the LRRK2 p.G2019S iPSCs and isogenic control cells were performed to identify novel molecular targets involved in LRRK2-parkinsonism pathways. RESULTS: ABE had a higher correction rate (13/53 clones, 24.5%) than HDR (3/47 clones, 6.4%). Twenty-seven HDR clones (57.4%), but no ABE clones, had deletions, though 14 ABE clones (26.4%) had off-target mutations. The corrected isogenic iPSC-derived dopaminergic neurons exhibited reduced LRRK2 kinase activity, decreased phospho-α-synuclein expression, and mitigated neurite shrinkage and apoptosis. Comparative transcriptomic and proteomic analysis identified different gene expression patterns in energy metabolism, protein degradation, and peroxisome proliferator-activated receptor pathways between the mutant and isogenic control cells. CONCLUSIONS: The results of this study envision that ABE could directly correct the pathogenic mutation in iPSCs for reversing disease-related phenotypes in neuropathology and exploring novel pathophysiological targets in PD.

Dynamic protein-RNA interactions in mediating splicing catalysis.

The spliceosome is assembled via sequential interactions of pre-mRNA with five small nuclear RNAs and many proteins. Recent determination of cryo-EM structures for several spliceosomal complexes has provided deep insights into interactions between spliceosomal components and structural changes of the spliceosome between steps, but information on how the proteins interact with pre-mRNA to mediate the reaction is scarce. By systematic analysis of proteins interacting with the splice sites (SSs), we have identified many previously unknown interactions of spliceosomal components with the pre-mRNA. Prp8 directly binds over the 5'SS and the branch site (BS) for the first catalytic step, and the 5'SS and 3'SS for the second step. Switching the Prp8 interaction from the BS to the 3'SS requires Slu7, which interacts dynamically with pre-mRNA first, and then interacts stably with the 3'-exon after Prp16-mediated spliceosome remodeling. Our results suggest that Prp8 plays a key role in positioning the 5'SS and 3'SS, facilitated by Slu7 through interactions with Prp8 and substrate RNA to advance exon ligation. We also provide evidence that Prp16 first docks on the intron 3' tail, then translocates in the 3' to 5' direction on remodeling the spliceosome.

The H/ACA complex disrupts triplex in hTR precursor to permit processing by RRP6 and PARN.

Human telomerase RNA (hTR) is transcribed as a precursor that is then posttranscriptionally modified and processed. A fraction of the transcripts is oligoadenylated by TRAMP and either processed into the mature hTR or degraded by the exosome. Here, we characterize the processing of 3' extended forms of varying length by PARN and RRP6. We show that tertiary RNA interactions unique to the longer transcripts favor RNA degradation, whereas H/ACA RNP assembly stimulates productive processing. Interestingly, the H/ACA complex actively promotes processing in addition to protecting the mature 3' end. Processing occurs in two steps with longer forms first being trimmed by RRP6 and shorter forms then being processed by PARN. These results reveal how RNA structure and RNP assembly affect the kinetics of processing and degradation and ultimately determine the amount of functional telomerase produced in cells.

A central role of Cwc25 in spliceosome dynamics during the catalytic phase of pre-mRNA splicing.

Splicing of precursor mRNA occurs via two consecutive steps of transesterification reaction; both require ATP and several proteins. Despite the energy requirement in the catalytic phase, incubation of the purified spliceosome under proper ionic conditions can elicit competitive reversible transesterification, debranching, and spliced-exon-reopening reactions without the necessity for ATP or other factors, suggesting that small changes in the conformational state of the spliceosome can lead to disparate chemical consequences for the substrate. We show here that Cwc25 plays a central role in modulating the conformational state of the catalytic spliceosome during normal splicing reactions. Cwc25 binds tightly to the spliceosome after the reaction and is then removed from the spliceosome, which normally requires DExD/H-box protein Prp16 and ATP hydrolysis, to allow the occurrence of the second reaction. When deprived of Cwc25, the purified first-step spliceosome catalyzes both forward and reverse splicing reactions under normal splicing conditions without requiring energy. Both reactions are inhibited when Cwc25 is added back, presumably due to the stabilization of first-step conformation. Prp16 is dispensable for the second reaction when splicing is carried out under conditions that destabilize Cwc25. We also show that the purified precatalytic spliceosome can catalyze two steps of the reaction at a low efficiency without requiring Cwc25, Slu7, or Prp18 when incubated under proper conditions. Our study reveals conformational modulation of the spliceosome by Cwc25 and Prp16 in stabilization and destabilization of first-step conformation, respectively, to facilitate the splicing process.

Human Telomerase RNA Processing and Quality Control.

The non-coding RNA subunit of telomerase provides the template for telomerase activity. In diverse fungi, 3' end processing of telomerase RNA involves a single cleavage by the spliceosome. Here, we examine how human telomerase RNA (hTR) primary transcripts are processed into the mature form of precisely 451 nt. We find that the splicing inhibitor isoginkgetin mimics the effects of RNA exosome inhibition and causes accumulation of long hTR transcripts. Depletion of exosome components and accessory factors reveals functions for the cap binding complex (CBC) and the nuclear exosome targeting (NEXT) complex in hTR turnover. Whereas longer transcripts are predominantly degraded, shorter precursor RNAs are oligo-adenylated by TRF4-2 and either processed by poly(A)-specific ribonuclease (PARN) or degraded by the exosome. Our results reveal that hTR biogenesis involves a kinetic competition between RNA processing and degradation and suggest treatment options for telomerase insufficiency disorders.

The spliceosome catalyzes debranching in competition with reverse of the first chemical reaction.

Splicing of nuclear pre-mRNA occurs via two steps of the transesterification reaction, forming a lariat intermediate and product. The reactions are catalyzed by the spliceosome, a large ribonucleoprotein complex composed of five small nuclear RNAs and numerous protein factors. The spliceosome shares a similar catalytic core structure with that of fungal group II introns, which can self-splice using the same chemical mechanism. Like group II introns, both catalytic steps of pre-mRNA splicing can efficiently reverse on the affinity-purified spliceosome. The spliceosome also catalyzes a hydrolytic spliced-exon reopening reaction as observed in group II introns, indicating a strong link in their evolutionary relationship. We show here that, by arresting splicing after the first catalytic step, the purified spliceosome can catalyze debranching of lariat-intron-exon 2. The debranching reaction, although not observed in group II introns, has similar monovalent cation preferences as those for splicing catalysis of group II introns. The debranching reaction is in competition with the reverse Step 1 reaction influenced by the ionic environment and the structure of components binding near the catalytic center, suggesting that the catalytic center of the spliceosome can switch between different conformations to direct different chemical reactions.

Link of NTR-mediated spliceosome disassembly with DEAH-box ATPases Prp2, Prp16, and Prp22.

The DEAH-box ATPase Prp43 is required for disassembly of the spliceosome after the completion of splicing or after the discard of the spliceosome due to a splicing defect. Prp43 associates with Ntr1 and Ntr2 to form the NTR complex and is recruited to the spliceosome via the interaction of Ntr2 and U5 component Brr2. Ntr2 alone can bind to U5 and to the spliceosome. To understand how NTR might mediate the disassembly of spliceosome intermediates, we arrested the spliceosome at various stages of the assembly pathway and assessed its susceptibility to disassembly. We found that NTR could catalyze the disassembly of affinity-purified spliceosomes arrested specifically after the ATP-dependent action of DEAH-box ATPase Prp2, Prp16, or Prp22 but not at steps before the action of these ATPases or upon their binding to the spliceosome. These results link spliceosome disassembly to the functioning of splicing ATPases. Analysis of the binding of Ntr2 to each splicing complex has revealed that the presence of Prp16 and Slu7, which also interact with Brr2, has a negative impact on Ntr2 binding. Our study provides insights into the mechanism by which NTR can be recruited to the spliceosome to mediate the disassembly of spliceosome intermediates when the spliceosome pathway is retarded, while disassembly is prevented in normal reactions.

DEAH-box ATPase Prp16 has dual roles in remodeling of the spliceosome in catalytic steps.

The assembly of the spliceosome involves dynamic rearrangements of interactions between snRNAs, protein components, and the pre-mRNA substrate. DExD/H-box ATPases are required to mediate structural changes of the spliceosome, utilizing the energy of ATP hydrolysis. Two DExD/H-box ATPases are required for the catalytic steps of the splicing pathway, Prp2 for the first step and Prp16 for the second step, both belonging to the DEAH subgroup of the protein family. The detailed mechanism of their action was not well understood until recently, when Prp2 was shown to be required for the release of U2 components SF3a and SF3b, presumably to allow the binding of Cwc25 to promote the first transesterification reaction. We show here that Cwc25 and Yju2 are released after the reaction in Prp16- and ATP-dependent manners, possibly to allow for the binding of Prp22, Prp18, and Slu7 to promote the second catalytic reaction. The binding of Cwc25 to the spliceosome is destabilized by mutations at the branchpoint sequence, suggesting that Cwc25 may bind to the branch site. We also show that Prp16 has an ATP-independent role in the first catalytic step, in addition to its known role in the second step. In the absence of ATP, Prp16 stabilizes the binding of Cwc25 to the spliceosome formed with branchpoint mutated pre-mRNAs to facilitate their splicing. Our results uncovered novel functions of Prp16 in both catalytic steps, and provide mechanistic insights into splicing catalysis.

Cwc25 is a novel splicing factor required after Prp2 and Yju2 to facilitate the first catalytic reaction.

Cwc25 has previously been identified to associate with pre-mRNA splicing factor Cef1/Ntc85, a component of the Prp19-associated complex (nineteen complex, or NTC) involved in spliceosome activation. We show here that Cwc25 is neither tightly associated with NTC nor required for spliceosome activation but is required for the first catalytic reaction. The affinity-purified spliceosome formed in Cwc25-depleted extracts contained only pre-mRNA and could be chased into splicing intermediates upon the addition of recombinant Cwc25 in an ATP-independent manner, suggesting that Cwc25 functions in the final step of the first catalytic reaction after the action of Prp2. Yju2 and a heat-resistant factor of unknown identity, HP, have previously been shown to be required for the same step of the splicing pathway. Cwc25, although resistant to heat treatment, is not sufficient to replace the function of HP, indicating that another heat-resistant factor, which we named HP-X, is involved. The requirement of Cwc25 and HP-X for the first catalytic reaction could be partially compensated for when the affinity-purified spliceosome was incubated in the presence of low concentrations of Mn(2+). These results have implications for the possible roles of Cwc25 and HP-X in facilitating juxtaposition of the 5' splice site and the branch point during the first catalytic reaction.

Both catalytic steps of nuclear pre-mRNA splicing are reversible.

Nuclear pre-messenger RNA (pre-mRNA) splicing is an essential processing step for the production of mature mRNAs from most eukaryotic genes. Splicing is catalyzed by a large ribonucleoprotein complex, the spliceosome, which is composed of five small nuclear RNAs and more than 100 protein factors. Despite the complexity of the spliceosome, the chemistry of the splicing reaction is simple, consisting of two consecutive transesterification reactions. The presence of introns in spliceosomal RNAs of certain fungi has suggested that splicing may be reversible; however, this has never been demonstrated experimentally. By using affinity-purified spliceosomes, we have shown that both catalytic steps of splicing can be efficiently reversed under appropriate conditions. These results provide considerable insight into the catalytic flexibility of the spliceosome.

Dynamic interactions of Ntr1-Ntr2 with Prp43 and with U5 govern the recruitment of Prp43 to mediate spliceosome disassembly.

The Saccharomyces cerevisiae splicing factors Ntr1 (also known as Spp382) and Ntr2 form a stable complex and can further associate with DExD/H-box RNA helicase Prp43 to form a functional complex, termed the NTR complex, which catalyzes spliceosome disassembly. We show that Prp43 interacts with Ntr1-Ntr2 in a dynamic manner. The Ntr1-Ntr2 complex can also bind to the spliceosome first, before recruiting Prp43 to catalyze disassembly. Binding of Ntr1-Ntr2 or Prp43 does not require ATP, but disassembly of the spliceosome requires hydrolysis of ATP. The NTR complex also dynamically interacts with U5 snRNP. Ntr2 interacts with U5 component Brr2 and is essential for both interactions of NTR with U5 and with the spliceosome. Ntr2 alone can also bind to U5 and to the spliceosome, suggesting a role of Ntr2 in mediating the binding of NTR to the spliceosome through its interaction with U5. Our results demonstrate that dynamic interactions of NTR with U5, through the interaction of Ntr2 with Brr2, and interactions of Ntr1 and Prp43 govern the recruitment of Prp43 to the spliceosome to mediate spliceosome disassembly.

Spliceosome disassembly catalyzed by Prp43 and its associated components Ntr1 and Ntr2.

Two novel yeast splicing factors required for spliceosome disassembly have been identified. Ntr1 and Ntr2 (NineTeen complex-Related proteins) were identified for their weak association with components of the Prp19-associated complex. Unlike other Prp19-associated components, these two proteins were primarily associated with the intron-containing spliceosome during the splicing reaction. Extracts depleted of Ntr1 or Ntr2 exhibited full splicing activity, but accumulated large amounts of lariat-intron in the spliceosome after splicing, indicating that the normal function of the Prp19-associated complex in spliceosome activation was not affected, but spliceosome disassembly was hindered. Immunoprecipitation analysis revealed that Ntr1 and Ntr2 formed a stable complex with DExD/H-box RNA helicase Prp43 in the splicing extract. Ntr1 interacted with Prp43 through the N-terminal G-patch domain, with Ntr2 through a middle region, and with itself through the carboxyl half of the protein. The affinity-purified Ntr1-Ntr2-Prp43 complex could catalyze disassembly of the spliceosome in an ATP-dependent manner, separating U2, U5, U6, NTC (NineTeen Complex), and lariat-intron. This is the first demonstration of physical disassembly of the spliceosome, catalyzed by a complex containing a DExD/H-box RNA helicase and two accessory factors, which might function in targeting the helicase to the correct substrate.