Obesity Drives STAT-1-Dependent NASH and STAT-3-Dependent HCC.

Obesity is a major driver of cancer, especially hepatocellular carcinoma (HCC). The prevailing view is that non-alcoholic steatohepatitis (NASH) and fibrosis or cirrhosis are required for HCC in obesity. Here, we report that NASH and fibrosis and HCC in obesity can be dissociated. We show that the oxidative hepatic environment in obesity inactivates the STAT-1 and STAT-3 phosphatase T cell protein tyrosine phosphatase (TCPTP) and increases STAT-1 and STAT-3 signaling. TCPTP deletion in hepatocytes promoted T cell recruitment and ensuing NASH and fibrosis as well as HCC in obese C57BL/6 mice that normally do not develop NASH and fibrosis or HCC. Attenuating the enhanced STAT-1 signaling prevented T cell recruitment and NASH and fibrosis but did not prevent HCC. By contrast, correcting STAT-3 signaling prevented HCC without affecting NASH and fibrosis. TCPTP-deletion in hepatocytes also markedly accelerated HCC in mice treated with a chemical carcinogen that promotes HCC without NASH and fibrosis. Our studies reveal how obesity-associated hepatic oxidative stress can independently contribute to the pathogenesis of NASH, fibrosis, and HCC.

Targeting of Polycomb Repressive Complex 2 to RNA by Short Repeats of Consecutive Guanines.

Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that trimethylates H3K27, a mark of repressed chromatin. Mammalian PRC2 binds RNA promiscuously, with thousands of target transcripts in vivo. But what does PRC2 recognize in these RNAs? Here we show that purified human PRC2 recognizes G > C,U ≫ A in single-stranded RNA and has a high affinity for folded guanine quadruplex (G4) structures but little binding to duplex RNAs. Importantly, G-tract motifs are significantly enriched among PRC2-binding transcripts in vivo. DNA sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on chromatin and H3K27me3-modified nucleosomes. Collectively, the abundance of PRC2-binding RNA motifs rationalizes the promiscuous RNA binding of PRC2, and their enrichment at Polycomb target genes provides a means for RNA-mediated regulation.

An investigation into the adsorption mechanism of n-butanol by ZIF-8: a combined experimental and ab initio molecular dynamics approach.

The zeolitic imidazolate framework, ZIF-8, has been shown by experimental methods to have a maximum saturation adsorption capacity of 0.36 g g-1 for n-butanol from aqueous solution, equivalent to a loading of 14 butanol molecules per unit cell or 7 molecules per sodalite ß-cage. Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) shows the presence of hydrogen bonding between adsorbed butanol molecules within the cage; the presence of three different O-H stretching modes indicates the formation of butanol clusters of varying size. Ab initio molecular dynamics simulations show the formation of intermolecular hydrogen bonding between the butanol molecules, with an average hydrogen-bond coordination number of 0.9 after 15 ps simulation time. The simulations also uniquely demonstrate the presence of weaker interactions between the alcohol O-H group and the π-orbital of the imidazole ring on the internal surface of the cage during early stages of adsorption. The calculated adsorption energy per butanol molecule is -33.7 kJ mol-1, confirming that the butanol is only weakly bound, driven primarily by the hydrogen bonding. Solid-state MAS NMR spectra suggest that the adsorbed butanol molecules possess a reasonable degree of mobility in their adsorbed state, rather than being rigidly held in specific sites. 2D 13C-1H heteronuclear correlation (HETCOR) experiments show interactions between the butanol aliphatic chain and the ZIF-8 framework experimentally, suggesting that O-H interactions with the π-orbital are only short lived. The insight gained from these results will allow the design of more efficient ways of recovering and isolating n-butanol, an important biofuel, from low-concentration solutions.

Modeling colorectal cancer: A bio-resource of 50 patient-derived organoid lines.

BACKGROUND AND AIM: Colorectal cancer (CRC) is the second leading cause of cancer death worldwide. To improve outcomes for these patients, we need to develop new treatment strategies. Personalized cancer medicine, where patients are treated based on the characteristics of their own tumor, has gained significant interest for its promise to improve outcomes and reduce unnecessary side effects. The purpose of this study was to examine the potential utility of patient-derived colorectal cancer organoids (PDCOs) in a personalized cancer medicine setting. METHODS: Patient-derived colorectal cancer organoids were derived from tissue obtained from treatment-naïve patients undergoing surgical resection for the treatment of CRC. We examined the recapitulation of key histopathological, molecular, and phenotypic characteristics of the primary tumor. RESULTS: We created a bio-resource of PDCOs from primary and metastatic CRCs. Key histopathological features were retained in PDCOs when compared with the primary tumor. Additionally, a cohort of 12 PDCOs, and their corresponding primary tumors and normal sample, were characterized through whole exome sequencing and somatic variant calling. These PDCOs exhibited a high level of concordance in key driver mutations when compared with the primary tumor. CONCLUSIONS: Patient-derived colorectal cancer organoids recapitulate characteristics of the tissue from which they are derived and are a powerful tool for cancer research. Further research will determine their utility for predicting patient outcomes in a personalized cancer medicine setting.

Dynamics of ribosome scanning and recycling revealed by translation complex profiling.

Regulation of messenger RNA translation is central to eukaryotic gene expression control. Regulatory inputs are specified by them RNA untranslated regions (UTRs) and often target translation initiation. Initiation involves binding of the 40S ribosomal small subunit (SSU) and associated eukaryotic initiation factors (eIFs)near the mRNA 5' cap; the SSU then scans in the 3' direction until it detects the start codon and is joined by the 60S ribosomal large subunit (LSU) to form the 80S ribosome. Scanning and other dynamic aspects of the initiation model have remained as conjectures because methods to trap early intermediates were lacking. Here we uncover the dynamics of the complete translation cycle in live yeast cells using translation complex profile sequencing (TCP-seq), a method developed from the ribosome profiling approach. We document scanning by observing SSU footprints along 5' UTRs. Scanning SSU have 5'-extended footprints (up to~75 nucleotides), indicative of additional interactions with mRNA emerging from the exit channel, promoting forward movement. We visualized changes in initiation complex conformation as SSU footprints coalesced into three major sizes at start codons (19, 29 and 37 nucleotides). These share the same 5' start site but differ at the 3' end, reflecting successive changes at the entry channel from an open to a closed state following start codon recognition. We also observe SSU 'lingering' at stop codons after LSU departure. Our results underpin mechanistic models of translation initiation and termination, built on decades of biochemical and structural investigation, with direct genome-wide in vivo evidence. Our approach captures ribosomal complexes at all phases of translation and will aid in studying translation dynamics in diverse cellular contexts. Dysregulation of translation is common in disease and, for example, SSU scanning is a target of anti-cancer drug development. TCP-seq will prove useful in discerning differences in mRNA-specific initiation in pathologies and their response to treatment.

A Dinuclear Osmium(II) Complex Near-Infrared Nanoscopy Probe for Nuclear DNA.

With the aim of developing photostable near-infrared cell imaging probes, a convenient route to the synthesis of heteroleptic OsII complexes containing the Os(TAP)2 fragment is reported. This method was used to synthesize the dinuclear OsII complex, [{Os(TAP)2}2tpphz]4+ (where tpphz = tetrapyrido[3,2-a:2',3'-c:3″,2''-h:2‴,3'''-j]phenazine and TAP = 1,4,5,8- tetraazaphenanthrene). Using a combination of resonance Raman and time-resolved absorption spectroscopy, as well as computational studies, the excited state dynamics of the new complex were dissected. These studies revealed that, although the complex has several close lying excited states, its near-infrared, NIR, emission (λmax = 780 nm) is due to a low-lying Os → TAP based 3MCLT state. Cell-based studies revealed that unlike its RuII analogue, the new complex is neither cytotoxic nor photocytotoxic. However, as it is highly photostable as well as live-cell permeant and displays NIR luminescence within the biological optical window, its properties make it an ideal probe for optical microscopy, demonstrated by its use as a super-resolution NIR STED probe for nuclear DNA.

Functional interplay between DEAD-box RNA helicases Ded1 and Dbp1 in preinitiation complex attachment and scanning on structured mRNAs in vivo.

RNA structures that impede ribosome binding or subsequent scanning of the 5'-untranslated region (5'-UTR) for the AUG initiation codon reduce translation efficiency. Yeast DEAD-box RNA helicase Ded1 appears to promote translation by resolving 5'-UTR structures, but whether its paralog, Dbp1, performs similar functions is unknown. Furthermore, direct in vivo evidence was lacking that Ded1 or Dbp1 resolves 5'-UTR structures that impede attachment of the 43S preinitiation complex (PIC) or scanning. Here, profiling of translating 80S ribosomes reveals that the translational efficiencies of many more mRNAs are reduced in a ded1-ts dbp1Δ double mutant versus either single mutant, becoming highly dependent on Dbp1 or Ded1 only when the other helicase is impaired. Such 'conditionally hyperdependent' mRNAs contain unusually long 5'-UTRs with heightened propensity for secondary structure and longer transcript lengths. Consistently, overexpressing Dbp1 in ded1 cells improves the translation of many such Ded1-hyperdependent mRNAs. Importantly, Dbp1 mimics Ded1 in conferring greater acceleration of 48S PIC assembly in a purified system on mRNAs harboring structured 5'-UTRs. Profiling 40S initiation complexes in ded1 and dbp1 mutants provides direct evidence that Ded1 and Dbp1 cooperate to stimulate both PIC attachment and scanning on many Ded1/Dbp1-hyperdependent mRNAs in vivo.

A Dinuclear Ruthenium(II) Complex Excited by Near-Infrared Light through Two-Photon Absorption Induces Phototoxicity Deep within Hypoxic Regions of Melanoma Cancer Spheroids.

The dinuclear photo-oxidizing RuII complex [{Ru(TAP2)}2(tpphz)]4+ (TAP = 1,4,5,8- tetraazaphenanthrene, tpphz = tetrapyrido[3,2-a:2',3'-c:3″,2''-h:2‴,3'''-j]phenazine), 14+, is readily taken up by live cells localizing in mitochondria and nuclei. In this study, the two-photon absorption cross section of 14+ is quantified and its use as a two-photon absorbing phototherapeutic is reported. It was confirmed that the complex is readily photoexcited using near-infrared, NIR, and light through two-photon absorption, TPA. In 2-D cell cultures, irradiation with NIR light at low power results in precisely focused phototoxicity effects in which human melanoma cells were killed after 5 min of light exposure. Similar experiments were then carried out in human cancer spheroids that provide a realistic tumor model for the development of therapeutics and phototherapeutics. Using the characteristic emission of the complex as a probe, its uptake into 280 µm spheroids was investigated and confirmed that the spheroid takes up the complex. Notably TPA excitation results in more intense luminescence being observed throughout the depth of the spheroids, although emission intensity still drops off toward the necrotic core. As 14+ can directly photo-oxidize DNA without the mediation of singlet oxygen or other reactive oxygen species, phototoxicity within the deeper, hypoxic layers of the spheroids was also investigated. To quantify the penetration of these phototoxic effects, 14+ was photoexcited through TPA at a power of 60 mW, which was progressively focused in 10 µm steps throughout the entire z-axis of individual spheroids. These experiments revealed that, in irradiated spheroids treated with 14+, acute and rapid photoinduced cell death was observed throughout their depth, including the hypoxic region.

Making the Right Link to Theranostics: The Photophysical and Biological Properties of Dinuclear RuII-ReI dppz Complexes Depend on Their Tether.

The synthesis of new dinuclear complexes containing linked RuII(dppz) and ReI(dppz) moieties is reported. The photophysical and biological properties of the new complex, which incorporates a N,N'-bis(4-pyridylmethyl)-1,6-hexanediamine tether ligand, are compared to a previously reported RuII/ReI complex linked by a simple dipyridyl alkane ligand. Although both complexes bind to DNA with similar affinities, steady-state and time-resolved photophysical studies reveal that the nature of the linker affects the excited state dynamics of the complexes and their DNA photocleavage properties. Quantum-based DFT calculations on these systems offer insights into these effects. While both complexes are live cells permeant, their intracellular localizations are significantly affected by the nature of the linker. Notably, one of the complexes displayed concentration-dependent localization and possesses photophysical properties that are compatible with SIM and STED nanoscopy. This allowed the dynamics of its intracellular localization to be tracked at super resolutions.

αß T-cell receptors with a central CDR3 cysteine are enriched in CD8αα intraepithelial lymphocytes and their thymic precursors.

The thymus plays a crucial role in immune tolerance by exposing developing T cells (thymocytes) to a myriad of self-antigens. Strong T-cell receptor (TCR) engagement induces tolerance in self-reactive thymocytes by stimulating apoptosis or selection into specialized T-cell lineages, including intestinal TCRαß+ CD8αα+ intraepithelial lymphocytes (IEL). TCR-intrinsic amino acid motifs that can be used to predict whether a TCR will be strongly self-reactive remain elusive. Here, a novel TCR sequence alignment approach revealed that T-cell lineages in C57BL/6 mice had divergent usage of cysteine within two positions of the amino acid at the apex of the complementarity-determining region 3 (CDR3) of the TCRα or TCRß chain. Compared to pre-selection thymocytes, central CDR3 cysteine usage was increased in IEL and Type A IEL precursors (IELp) and markedly decreased in Foxp3+ regulatory T cells (T-reg) and naïve T cells. These findings reveal a TCR-intrinsic motif that distinguishes Type A IELp and IEL from T-reg and naïve T cells.

Identification of RNA-binding Proteins in Macrophages by Interactome Capture.

Pathogen components, such as lipopolysaccharides of Gram-negative bacteria that activate Toll-like receptor 4, induce mitogen activated protein kinases and NFκB through different downstream pathways to stimulate pro- and anti-inflammatory cytokine expression. Importantly, post-transcriptional control of the expression of Toll-like receptor 4 downstream signaling molecules contributes to the tight regulation of inflammatory cytokine synthesis in macrophages. Emerging evidence highlights the role of RNA-binding proteins (RBPs) in the post-transcriptional control of the innate immune response. To systematically identify macrophage RBPs and their response to LPS stimulation, we employed RNA interactome capture in LPS-induced and untreated murine RAW 264.7 macrophages. This combines RBP-crosslinking to RNA, cell lysis, oligo(dT) capture of polyadenylated RNAs and mass spectrometry analysis of associated proteins. Our data revealed 402 proteins of the macrophage RNA interactome including 91 previously not annotated as RBPs. A comparison with published RNA interactomes classified 32 RBPs uniquely identified in RAW 264.7 macrophages. Of these, 19 proteins are linked to biochemical activities not directly related to RNA. From this group, we validated the HSP90 cochaperone P23 that was demonstrated to exhibit cytosolic prostaglandin E2 synthase 3 (PTGES3) activity, and the hematopoietic cell-specific LYN substrate 1 (HCLS1 or HS1), a hematopoietic cell-specific adapter molecule, as novel macrophage RBPs. Our study expands the mammalian RBP repertoire, and identifies macrophage RBPs that respond to LPS. These RBPs are prime candidates for the post-transcriptional regulation and execution of LPS-induced signaling pathways and the innate immune response. Macrophage RBP data have been deposited to ProteomeXchange with identifier PXD002890.

Directly Coupled Versus Spectator Linkers on Diimine PtII Acetylides-Change the Structure, Keep the Function?

Modification of light-harvesting units with anchoring groups for surface attachment often compromises light-harnessing properties. Herein, a series of [donor-acceptor-anchor] platinum(II) diimine (bis-)acetylides was developed in order to systematically compare the effect of conjugated versus electronically decoupled modes of attachment of protected anchoring groups on the photophysical properties of light-harvesting units. The first examples of "decoupled" phosphonate diimine PtII complexes are reported, and their properties are compared and contrasted to those of carboxylate analogues studied by a diversity of methods. Ultrafast time-resolved IR and transient absorption spectroscopy revealed that all complexes have a charge-transfer (CT) lowest excited state with lifetimes between 2 and 14 ns. Vibrational signatures and dynamics of CT states were identified; the assignment of electronic states and their vibrational origin was aided by TDDFT calculations. Ultrafast energy redistribution accompanied by structural changes was directly captured in the CT states. A significant difference between the structures of the electronic ground and CT excited states, as well as differences in the structural reorganisation in the complexes bearing directly attached or electronically decoupled anchoring groups, was discovered. This work demonstrates that decoupling of the anchoring group from the light-harvesting core by a saturated spacer is an easy approach to combine surface attachment with high reduction potential and ten times longer lifetime of the CT excited state of the light-absorbing unit, and retain electron-transfer photoreactivity essential for light-harvesting applications.

Homo- and Heteroleptic Phototoxic Dinuclear Metallo-Intercalators Based on RuII (dppn) Intercalating Moieties: Synthesis, Optical, and Biological Studies.

Using a new mononuclear "building block," for the first time, a dinuclear RuII (dppn) complex and a heteroleptic system containing both RuII (dppz) and RuII (dppn) moieties are reported. The complexes, including the mixed dppz/dppn system, are 1 O2 sensitizers. However, unlike the homoleptic dppn systems, the mixed dppz/dppn complex also displays a luminescence "switch on" DNA light-switch effect. In both cisplatin sensitive and resistant human ovarian carcinoma lines the dinuclear complexes show enhanced uptake compared to their mononuclear analogue. Thanks to a favorable combination of singlet oxygen generation and cellular uptake properties all three of the new complexes are phototoxic and display potent activity against chemotherapeutically resistant cells.

(13)C or Not (13)C: Selective Synthesis of Asymmetric Carbon-13-Labeled Platinum(II) cis-Acetylides.

Asymmetric isotopic labeling of parallel and identical electron- or energy-transfer pathways in symmetrical molecular assemblies is an extremely challenging task owing to the inherent lack of isotopic selectivity in conventional synthetic methods. Yet, it would be a highly valuable tool in the study and control of complex light-matter interactions in molecular systems by exclusively and nonintrusively labeling one of otherwise identical reaction pathways, potentially directing charge and energy transport along a chosen path. Here we describe the first selective synthetic route to asymmetrically labeled organometallic compounds, on the example of charge-transfer platinum(II) cis-acetylide complexes. We demonstrate the selective (13)C labeling of one of two acetylide groups. We further show that such isotopic labeling successfully decouples the two ν(C≡C) in the mid-IR region, permitting independent spectroscopic monitoring of two otherwise identical electron-transfer pathways, along the (12)C≡(12)C and (13)C≡(13)C coordinates. Quantum-mechanical mixing leads to intriguing complex features in the vibrational spectra of such species, which we successfully model by full-dimensional anharmonically corrected DFT calculations, despite the large size of these systems. The synthetic route developed and demonstrated herein should lead to a great diversity of asymmetric organometallic complexes inaccessible otherwise, opening up a plethora of opportunities to advance the fundamental understanding and control of light-matter interactions in molecular systems.

Probing the closed-loop model of mRNA translation in living cells.

The mRNA closed-loop, formed through interactions between the cap structure, poly(A) tail, eIF4E, eIF4G and PAB, features centrally in models of eukaryotic translation initiation, although direct support for its existence in vivo is not well established. Here, we investigated the closed-loop using a combination of mRNP isolation from rapidly cross-linked cells and high-throughput qPCR. Using the interaction between these factors and the opposing ends of mRNAs as a proxy for the closed-loop, we provide evidence that it is prevalent for eIF4E/4G-bound but unexpectedly sparse for PAB1-bound mRNAs, suggesting it primarily occurs during a distinct phase of polysome assembly. We observed mRNA-specific variation in the extent of closed-loop formation, consistent with a role for polysome topology in the control of gene expression.

Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage.

BACKGROUND: A major hurdle to transcriptome profiling by deep-sequencing technologies is that abundant transcripts, such as rRNAs, can overwhelm the libraries, severely reducing transcriptome-wide coverage. Methods for depletion of such unwanted sequences typically require treatment of RNA samples prior to library preparation, are costly and not suited to unusual species and applications. Here we describe Probe-Directed Degradation (PDD), an approach that employs hybridisation to DNA oligonucleotides at the single-stranded cDNA library stage and digestion with Duplex-Specific Nuclease (DSN). RESULTS: Targeting Saccharomyces cerevisiae rRNA sequences in Illumina HiSeq libraries generated by the split adapter method we show that PDD results in efficient removal of rRNA. The probes generate extended zones of depletion as a function of library insert size and the requirements for DSN cleavage. Using intact total RNA as starting material, probes can be spaced at the minimum anticipated library size minus 20 nucleotides to achieve continuous depletion. No off-target bias is detectable when comparing PDD-treated with untreated libraries. We further provide a bioinformatics tool to design suitable PDD probe sets. CONCLUSION: We find that PDD is a rapid procedure that results in effective and specific depletion of unwanted sequences from deep-sequencing libraries. Because PDD acts at the cDNA stage, handling of fragile RNA samples can be minimised and it should further be feasible to remediate existing libraries. Importantly, PDD preserves the original RNA fragment boundaries as is required for nucleotide-resolution footprinting or base-cleavage studies. Finally, as PDD utilises unmodified DNA oligonucleotides it can provide a low-cost option for large-scale projects, or be flexibly customised to suit different depletion targets, sample types and organisms.

A Retrospective Review and Comprehensive Tumour Profiling of Advanced Non-Melanomatous Cutaneous Spindle Cell Neoplasms Treated with Immune-Checkpoint Inhibitors.

Non-melanomatous cutaneous spindle cell neoplasms are a rare group of malignancies that present a diagnostic challenge, and for which there is a lack of consensus on how to best manage patients with advanced disease and only limited reports of immune-checkpoint inhibitor (ICI) responses. In this study, we performed a single-center retrospective review of treatment outcomes for all advanced non-melanomatous cutaneous spindle cell neoplasms treated with ICIs. Blinded histopathology reviews occurred to confirm each diagnosis. Comprehensive tumour profiling included whole exome sequencing for tumour mutational burden (TMB) and ultraviolet(UV) signatures, and immunohistochemistry for immune-cell infiltration (CD4/CD3/CD8/CD103/CD20) and immune-checkpoint expression (PD-L1/LAG3/TIGIT). Seven patients were identified. The objective response rate was 86% (6/7) with five complete responses (CR). Responses were durable with two patients in CR > 30 months after ICI commencement. All patients had high TMB and UV signatures. One patient had PD-L1 100% (combined positive score) with abundant immune-cell infiltration and LAG3 expression. In advanced non-melanomatous cutaneous spindle cell neoplasms, excellent responses to ICIs with durable disease control were observed. ICIs are worthy of further exploration in these patients. UV signatures and high TMB could be used to help select patients for treatment.

Selectively inhibiting malignant melanoma migration and invasion in an engineered skin model using actin-targeting dinuclear RuII-complexes.

Due to the poor prognosis of metastatic cancers, there is a clinical need for agents with anti-metastatic activity. Here we report on the anti-metastatic effect of a previously reported Ru(ii) complex [{(phen)2Ru}2(tpphz)]4+, 14+, that has recently been shown to disrupt actin fiber assembly. In this study, we investigated the anti-migratory effect of +14+ and a close structural analogue+, 24+, on two highly invasive, metastatic human melanoma cell lines. Laser scanning confocal imaging was used to investigate the structure of actin filament and adhesion molecule vinculin and results show disassembly of central actin filaments and focal adhesions. The effect of both compounds on actin filaments was also found to be reversible. As these results revealed that the complexes were cytostatic and produced a significant inhibitory effect on the migration of both melanoma cell lines but not human dermal fibroblasts their effect on 3D-spheroids and a tissue-engineered living skin model were also investigated. These experiments demonstrated that the compounds inhibited the growth and invasiveness of the melanoma-based spheroidal tumor model and both complexes were found to penetrate the epidermis of the skin tissue model and inhibit the invasion of melanoma cells. Taken together, the cytostatic and antimigratory effects of the complexes results in an antimetastatic effect that totally prevent invasion of malignant melanoma into skin tissue.

Functional recovery of the germ line following splicing collapse.

Splicing introns from precursor-messenger RNA (pre-mRNA) transcripts is essential for translating functional proteins. Here, we report that the previously uncharacterized Caenorhabditis elegans protein MOG-7 acts as a pre-mRNA splicing factor. Depleting MOG-7 from the C. elegans germ line causes intron retention in most germline-expressed genes, impeding the germ cell cycle, and causing defects in nuclear morphology, germ cell identity and sterility. Despite the deleterious consequences caused by MOG-7 loss, the adult germ line can functionally recover to produce viable and fertile progeny when MOG-7 is restored. Germline recovery is dependent on a burst of apoptosis that likely clears defective germ cells, and viable gametes generated from the proliferation of germ cells in the progenitor zone. Together, these findings reveal that MOG-7 is essential for germ cell development, and that the germ line can functionally recover after a collapse in RNA splicing.