The E592K variant of SF3B1 creates unique RNA missplicing and associates with high-risk MDS without ring sideroblasts.

Among the most common genetic alterations in the myelodysplastic syndromes (MDS) are mutations in the spliceosome gene SF3B1. Such mutations induce specific RNA missplicing events, directly promote ring sideroblast (RS) formation, and generally associate with more favorable prognosis. However, not all SF3B1 mutations are the same, and little is known about how distinct hotspots influence disease. Here we report that the E592K variant of SF3B1 associates with high-risk disease features in MDS, including a lack of RS, increased myeloblasts, a distinct co-mutation pattern, and a lack of the favorable survival seen with other SF3B1 mutations. Moreover, compared to other hotspot SF3B1 mutations, E592K induces a unique RNA missplicing pattern, retains an interaction with the splicing factor SUGP1, and preserves normal RNA splicing of the sideroblastic anemia genes TMEM14C and ABCB7. These data have implications for our understanding of the functional diversity of spliceosome mutations, as well as the pathobiology, classification, prognosis, and management of SF3B1-mutant MDS.

Non-canonical isoforms of the mRNA polyadenylation factor WDR33 regulate STING-mediated immune responses.

The human WDR33 gene encodes three major isoforms. The canonical isoform WDR33v1 (V1) is a well-characterized nuclear mRNA polyadenylation factor, while the other two, WDR33v2 (V2) and WDR33v3 (V3), have not been studied. Here, we report that V2 and V3 are generated by alternative polyadenylation, and neither protein contains all seven WD (tryptophan-aspartic acid) repeats that characterize V1. Surprisingly, V2 and V3 are not polyadenylation factors but localize to the endoplasmic reticulum and interact with stimulator of interferon genes (STING), the immune factor that induces the cellular response to cytosolic double-stranded DNA. V2 suppresses interferon-ß induction by preventing STING disulfide oligomerization but promotes autophagy, likely by recruiting WIPI2 isoforms. V3, on the other hand, functions to increase STING protein levels. Our study has not only provided mechanistic insights into STING regulation but also revealed that protein isoforms can be functionally completely unrelated, indicating that alternative mRNA processing is a more powerful mechanism than previously appreciated.

Characterization of the SF3B1-SUGP1 interface reveals how numerous cancer mutations cause mRNA missplicing.

The spliceosomal gene SF3B1 is frequently mutated in cancer. While it is known that SF3B1 hotspot mutations lead to loss of splicing factor SUGP1 from spliceosomes, the cancer-relevant SF3B1-SUGP1 interaction has not been characterized. To address this issue, we show by structural modeling that two regions flanking the SUGP1 G-patch make numerous contacts with the region of SF3B1 harboring hotspot mutations. Experiments confirmed that all the cancer-associated mutations in these regions, as well as mutations affecting other residues in the SF3B1-SUGP1 interface, not only weaken or disrupt the interaction but also alter splicing similarly to SF3B1 cancer mutations. Finally, structural modeling of a trimeric protein complex reveals that the SF3B1-SUGP1 interaction "loops out" the G-patch for interaction with the helicase DHX15. Our study thus provides an unprecedented molecular view of a protein complex essential for accurate splicing and also reveals that numerous cancer-associated mutations disrupt the critical SF3B1-SUGP1 interaction.

The E592K variant of SF3B1 creates unique RNA missplicing and associates with high-risk MDS without ring sideroblasts.

Among the most common genetic alterations in the myelodysplastic syndromes (MDS) are mutations in the spliceosome gene SF3B1. Such mutations induce specific RNA missplicing events, directly promote ring sideroblast (RS) formation, generally associate with more favorable prognosis, and serve as a predictive biomarker of response to luspatercept. However, not all SF3B1 mutations are the same, and here we report that the E592K variant of SF3B1 associates with high-risk disease features in MDS, including a lack of RS, increased myeloblasts, a distinct co-mutation pattern, and decreased survival. Moreover, in contrast to canonical SF3B1 mutations, E592K induces a unique RNA missplicing pattern, retains an interaction with the splicing factor SUGP1, and preserves normal RNA splicing of the sideroblastic anemia genes TMEM14C and ABCB7. These data expand our knowledge of the functional diversity of spliceosome mutations, and they suggest that patients with E592K should be approached differently from low-risk, luspatercept-responsive MDS patients with ring sideroblasts and canonical SF3B1 mutations.

A nucleolar long "non-coding" RNA encodes a novel protein that functions in response to stress.

Certain long non-coding RNAs (lncRNAs) are known to contain small open reading frames that can be translated. Here we describe a much larger 25 kDa human protein, "Ribosomal IGS Encoded Protein" (RIEP), that remarkably is encoded by the well-characterized RNA polymerase (RNAP) II-transcribed nucleolar "promoter and pre-rRNA antisense" lncRNA (PAPAS). Strikingly, RIEP, which is conserved throughout primates but not found in other species, predominantly localizes to the nucleolus as well as mitochondria, but both exogenously expressed and endogenous RIEP increase in the nuclear and perinuclear regions upon heat shock (HS). RIEP associates specifically with the rDNA locus, increases levels of the RNA:DNA helicase Senataxin, and functions to sharply reduce DNA damage induced by heat shock. Proteomics analysis identified two mitochondrial proteins, C1QBP and CHCHD2, both known to have mitochondrial and nuclear functions, that we show interact directly, and relocalize following heat shock, with RIEP. Finally, it is especially notable that the rDNA sequences encoding RIEP are multifunctional, giving rise to an RNA that functions both as RIEP messenger RNA (mRNA) and as PAPAS lncRNA, as well as containing the promoter sequences responsible for rRNA synthesis by RNAP I. Our work has thus not only shown that a nucleolar "non-coding" RNA in fact encodes a protein, but also established a novel link between mitochondria and nucleoli that contributes to the cellular stress response.

DHX15 is involved in SUGP1-mediated RNA missplicing by mutant SF3B1 in cancer.

SF3B1 is the most frequently mutated spliceosomal gene in cancer. Several hotspot mutations are known to disrupt the interaction of SF3B1 with another splicing factor, SUGP1, resulting in the RNA missplicing that characterizes mutant SF3B1 cancers. Properties of SUGP1, especially the presence of a G-patch motif, a structure known to function by activating DEAH-box RNA helicases, suggest the requirement of such an enzyme in SUGP1 function in splicing. However, the identity of this putative helicase has remained an important unanswered question. Here, using a variety of protein-protein interaction assays, we identify DHX15 as the critical helicase. We further show that depletion of DHX15 or expression of any of several DHX15 mutants, including one implicated in acute myeloid leukemia, partially recapitulates the splicing defects of mutant SF3B1. Moreover, a DHX15-SUGP1 G-patch fusion protein is able to incorporate into the spliceosome to rescue the splicing defects of mutant SF3B1. We also present the crystal structure of the human DHX15-SUGP1 G-patch complex, which reveals the molecular basis of their direct interaction. Our data thus demonstrate that DHX15 is the RNA helicase that functions with SUGP1 and additionally provide important insight into how mutant SF3B1 disrupts splicing in cancer.

Beyond rRNA: nucleolar transcription generates a complex network of RNAs with multiple roles in maintaining cellular homeostasis.

Nucleoli are the major cellular compartments for the synthesis of rRNA and assembly of ribosomes, the macromolecular complexes responsible for protein synthesis. Given the abundance of ribosomes, there is a huge demand for rRNA, which indeed constitutes ∼80% of the mass of RNA in the cell. Thus, nucleoli are characterized by extensive transcription of multiple rDNA loci by the dedicated polymerase, RNA polymerase (Pol) I. However, in addition to producing rRNAs, there is considerable additional transcription in nucleoli by RNA Pol II as well as Pol I, producing multiple noncoding (nc) and, in one instance, coding RNAs. In this review, we discuss important features of these transcripts, which often appear species-specific and reflect transcription antisense to pre-rRNA by Pol II and within the intergenic spacer regions on both strands by both Pol I and Pol II. We discuss how expression of these RNAs is regulated, their propensity to form cotranscriptional R loops, and how they modulate rRNA transcription, nucleolar structure, and cellular homeostasis more generally.

Relationship of cash transfers with risk of overweight and obesity in children and adults: a systematic review.

BACKGROUND: Cash transfer (CT) programs are an important type of social protection meant to reduce poverty. Whether CT programs increase the risk of overweight and obesity is unclear. The objective was to characterize the relationship between CT programs and the risk of overweight and obesity in children and adults. METHODS: We searched articles in PubMed, Embase, Cochrane, EconLit, Global Health, CINAHL Plus, IBSS, Health & Medical Collection, Scopus, Web of Science, and WHO Global Index Medicus in August 2021. Studies involving CT as the intervention, a control group, body mass index, overweight, or obesity as an outcome, and sample size > 300 were included. The Newcastle-Ottawa Scale was used for quality assessment. RESULTS: Of 2355 articles identified, 20 met the inclusion criteria. Because of marked heterogeneity in methodology, a narrative synthesis was used to present results. Thirteen of the studies reported that CT programs were associated with a significantly lower risk of overweight and obesity, eight studies showed no significant association, and one study reported a significantly increased risk of obesity in women. Quality assessment showed that most studies lacked sample size and power calculations, validation of exposure, descriptions of non-respondents or those lost to follow-up, and blinded outcome assessment. CONCLUSIONS: Overall, the studies were suggestive that CT programs either have no impact or decrease the risk of overweight and/or obesity in children, adolescents, and adults, but no firm conclusions can be drawn from the available evidence. This review demonstrated limitations in the available studies of CT programs and overweight/obesity.

Nuclear RNA transcript levels modulate nucleocytoplasmic distribution of ALS/FTD-associated protein FUS.

Fused in Sarcoma (FUS) is a nuclear RNA/DNA binding protein that mislocalizes to the cytoplasm in the neurodegenerative diseases ALS and FTD. Despite the existence of FUS pathogenic mutations that result in nuclear import defects, a subset of ALS/FTD patients display cytoplasmic accumulation of wild-type FUS, although the underlying mechanism is unclear. Here we confirm that transcriptional inhibition, specifically of RNA polymerase II (RNAP II), induces FUS cytoplasmic translocation, but we show that several other stresses do not. We found unexpectedly that the epitope specificity of different FUS antibodies significantly affects the apparent FUS nucleocytoplasmic ratio as determined by immunofluorescence, explaining inconsistent observations in previous studies. Significantly, depletion of the nuclear mRNA export factor NXF1 or RNA exosome cofactor MTR4 promotes FUS nuclear retention, even when transcription is repressed, while mislocalization was independent of the nuclear protein export factor CRM1 and import factor TNPO1. Finally, we report that levels of nascent RNAP II transcripts, including those known to bind FUS, are reduced in sporadic ALS iPS cells, linking possible aberrant transcriptional control and FUS cytoplasmic mislocalization. Our findings thus reveal that factors that influence accumulation of nuclear RNAP II transcripts modulate FUS nucleocytoplasmic homeostasis, and provide evidence that reduced RNAP II transcription can contribute to FUS mislocalization to the cytoplasm in ALS.

More evidence on cash transfers and child nutritional outcomes: a systematic review and meta-analysis.

BACKGROUND: Cash transfer (CT) programmes are an increasingly common approach to alleviate poverty and inequality and improving child health and nutrition, as well as supporting other goals such as education. Evidence indicates that CTs can be effective, but overall impacts are small in magnitude. This paper substantially updates the evidence base on the effectiveness of CTs and moderating factors. METHODS: Building on a prior search done in 2018, we searched articles published between January 2018 and March 2021 using Agris, Econlit, Eldis, IBSS, IDEAS, IFPRI, Google Scholar, PubMed and World Bank databases. We included studies using quantitative impact evaluation methods of CTs with sample sizes over 300, targeted to households with children under 5 years conducted with clear counterfactuals in countries with gross domestic product below US$10 000 at baseline. We performed meta-analysis using random effects models to assess the impact of CT programme on selected child nutrition outcomes. FINDINGS: Out of 1561 articles identified, 55 additional articles were eligible for inclusion for a total of 129 estimates. We find that CTs have significant although modest effects on height-for-age z-scores (HAZ) (0.024, 95% CI 0.004 to 0.044; p<0.02); stunting (-1.35%, 95% CI -2.35 to - 0.35; p<0;01); wasting (-1.31%, 95% CI -2.16% to 0.46%; p<0.01); animal-source foods (6.72%, 95% CI 5.24% to 8.20%; p<0.01); diet diversity (0.55, 95% CI 0.30 to 0.81; p<0.01) and diarrhoea incidence (- 1.74%, 95% CI -2.79% to -0.68%; p<0.05). There was no significant effect of CTs on weight-for-height (WHZ) or weight-for-age z-scores (WAZ). Well-targeted behaviour change communication was also effective in improving HAZ and decreasing the prevalence of diarrhoea. INTERPRETATION: CT programmes improved linear growth among young children, reducing wasting and stunting, but effects are heterogeneous and somewhat small overall. More evidence indicates that effects on dietary diversity and the consumption of animal-source foods are increasingly pronounced.

SF3B1 mutant-induced missplicing of MAP3K7 causes anemia in myelodysplastic syndromes.

SF3B1 is the most frequently mutated RNA splicing factor in cancer, including in ∼25% of myelodysplastic syndromes (MDS) patients. SF3B1-mutated MDS, which is strongly associated with ringed sideroblast morphology, is characterized by ineffective erythropoiesis, leading to severe, often fatal anemia. However, functional evidence linking SF3B1 mutations to the anemia described in MDS patients harboring this genetic aberration is weak, and the underlying mechanism is completely unknown. Using isogenic SF3B1 WT and mutant cell lines, normal human CD34 cells, and MDS patient cells, we define a previously unrecognized role of the kinase MAP3K7, encoded by a known mutant SF3B1-targeted transcript, in controlling proper terminal erythroid differentiation, and show how MAP3K7 missplicing leads to the anemia characteristic of SF3B1-mutated MDS, although not to ringed sideroblast formation. We found that p38 MAPK is deactivated in SF3B1 mutant isogenic and patient cells and that MAP3K7 is an upstream positive effector of p38 MAPK. We demonstrate that disruption of this MAP3K7-p38 MAPK pathway leads to premature down-regulation of GATA1, a master regulator of erythroid differentiation, and that this is sufficient to trigger accelerated differentiation, erythroid hyperplasia, and ultimately apoptosis. Our findings thus define the mechanism leading to the severe anemia found in MDS patients harboring SF3B1 mutations.

Oxidative stress induces Ser 2 dephosphorylation of the RNA polymerase II CTD and premature transcription termination.

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) consists of YSPTSPS heptapeptide repeats, and the phosphorylation status of the repeats controls multiple transcriptional steps and co-transcriptional events. However, how CTD phosphorylation status responds to distinct environmental stresses is not fully understood. In this study, we found that a drastic reduction in phosphorylation of a subset of Ser2 residues occurs rapidly but transiently following exposure to H2O2. ChIP analysis indicated that Ser2-P, and to a lesser extent Tyr1-P was reduced only at the gene 3' end. Significantly, the levels of polyadenylation factor CstF77, as well as Pol II, were also reduced. However, no increase in uncleaved or readthrough RNA products was observed, suggesting transcribing Pol II prematurely terminates at the gene end in response to H2O2. Further analysis found that the reduction of Ser2-P is, at least in part, regulated by CK2 but independent of FCP1 and other known Ser2 phosphatases. Finally, the H2O2 treatment also affected snRNA 3' processing although surprisingly the U2 processing was not impaired. Together, our data suggest that H2O2 exposure creates a unique CTD phosphorylation state that rapidly alters transcription to deal with acute oxidative stress, perhaps creating a novel "emergency brake" mechanism to transiently dampen gene expression.

Replication protein A associates with nucleolar R loops and regulates rRNA transcription and nucleolar morphology.

The nucleolus is an important cellular compartment in which ribosomal RNAs (rRNAs) are transcribed and where certain stress pathways that are crucial for cell growth are coordinated. Here we report novel functions of the DNA replication and repair factor replication protein A (RPA) in control of nucleolar homeostasis. We show that loss of the DNA:RNA helicase senataxin (SETX) promotes RPA nucleolar localization, and that this relocalization is dependent on the presence of R loops. Notably, this nucleolar RPA phenotype was also observed in the presence of camptothecin (CPT)-induced genotoxic stress, as well as in SETX-deficient AOA2 patient fibroblasts. Extending these results, we found that RPA is recruited to rDNA following CPT treatment, where RPA prevents R-loop-induced DNA double-strand breaks. Furthermore, we show that loss of RPA significantly decreased 47S pre-rRNA levels, which was accompanied by increased expression of both RNAP II-mediated "promoter and pre-rRNA antisense" RNA as well as RNAP I-transcribed intragenic spacer RNAs. Finally, and likely reflecting the above, we found that loss of RPA promoted nucleolar structural disorganization, characterized by the appearance of reduced size nucleoli. Our findings both indicate new roles for RPA in nucleoli through pre-rRNA transcriptional control and also emphasize that RPA function in nucleolar homeostasis is linked to R-loop resolution under both physiological and pathological conditions.

Multiple ways to a dead end: diverse mechanisms by which ALS mutant genes induce cell death.

Amyotrophic Lateral Sclerosis (ALS) is a deadly neuromuscular disorder caused by progressive motor neuron loss in the brain and spinal cord. Over the past decades, a number of genetic mutations have been identified that cause or are associated with ALS disease progression. Numerous genes harbor ALS mutations, and they encode proteins displaying a wide range of physiological functions, with limited overlap. Despite the divergent functions, mutations in these genes typically trigger protein aggregation, which can confer gain- and/or loss-of-function to a number of essential cellular processes. Nuclear processes such as mRNA splicing and the response to DNA damage are significantly affected in ALS patients. Cytoplasmic organelles such as mitochondria are damaged by ALS mutant proteins. Processes that maintain cellular homeostasis such as autophagy, nonsense-mediated mRNA decay and nucleocytoplasmic transport, are also impaired by ALS mutations. Here, we review the multiple mechanisms by which mutations in major ALS-associated genes, such as TARDBP, C9ORF72 and FUS, lead to impairment of essential cellular processes.

Coronavirus infections and deaths by poverty status: The effects of social distancing.

We study the spread of COVID-19 infections and deaths by county poverty level in the US. In the beginning of the pandemic, counties with either very low poverty levels or very high poverty levels reported the highest numbers of cases. A U-shaped relationship prevails for counties with high population density while among counties with low population density, only poorer counties report high incidence rates of COVID-19. Second, we discuss the pattern of infections spreading from higher to lower income counties. Third, we show that stay-at-home mandates caused significantly higher reductions in mobility in high income counties that experienced adverse weather shocks than counties that did not. These effects are not present in counties with high poverty rates. Using weather shocks in combination with stay-at-home mandates as an instrument for social distancing, we find that measures taken to promote social distancing helped curb infections in high income counties but not in low income counties. These results have important policy implications for containing the spread of infectious diseases in the future.

SETX (senataxin), the helicase mutated in AOA2 and ALS4, functions in autophagy regulation.

SETX (senataxin) is an RNA/DNA helicase that has been implicated in transcriptional regulation and the DNA damage response through resolution of R-loop structures. Mutations in SETX result in either of two distinct neurodegenerative disorders. SETX dominant mutations result in a juvenile form of amyotrophic lateral sclerosis (ALS) called ALS4, whereas recessive mutations are responsible for ataxia called ataxia with oculomotor apraxia type 2 (AOA2). How mutations in the same protein can lead to different phenotypes is still unclear. To elucidate AOA2 disease mechanisms, we first examined gene expression changes following SETX depletion. We observed the effects on both transcription and RNA processing, but surprisingly observed decreased R-loop accumulation in SETX-depleted cells. Importantly, we discovered a strong connection between SETX and the macroautophagy/autophagy pathway, reflecting a direct effect on transcription of autophagy genes. We show that SETX depletion inhibits the progression of autophagy, leading to an accumulation of ubiquitinated proteins, decreased ability to clear protein aggregates, as well as mitochondrial defects. Analysis of AOA2 patient fibroblasts also revealed a perturbation of the autophagy pathway. Our work has thus identified a novel function for SETX in the regulation of autophagy, whose modulation may have a therapeutic impact for AOA2.Abbreviations: 3'READS: 3' region extraction and deep sequencing; ACTB: actin beta; ALS4: amyotrophic lateral sclerosis type 4; AOA2: ataxia with oculomotor apraxia type 2; APA: alternative polyadenylation; AS: alternative splicing; ATG7: autophagy-related 7; ATP6V0D2: ATPase H+ transporting V0 subunit D2; BAF: bafilomycin A1; BECN1: beclin 1; ChIP: chromatin IP; Chloro: chloroquine; CPT: camptothecin; DDR: DNA damage response; DNMT1: DNA methyltransferase 1; DRIP: DNA/RNA IP; DSBs: double strand breaks; EBs: embryoid bodies; FTD: frontotemporal dementia; GABARAP: GABA type A receptor-associated protein; GO: gene ontology; HR: homologous recombination; HTT: huntingtin; IF: immunofluorescence; IP: immunoprecipitation; iPSCs: induced pluripotent stem cells; KD: knockdown; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MN: motor neuron; MTORC1: mechanistic target of rapamycin kinase complex 1; PASS: PolyA Site Supporting; PFA: paraformaldehyde; RNAPII: RNA polymerase II; SCA: spinocerebellar ataxia; SETX: senataxin; SMA: spinal muscular atrophy; SMN1: survival of motor neuron 1, telomeric; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TSS: transcription start site; TTS: transcription termination site; ULK1: unc-51 like autophagy activating kinase 1; WB: western blot; WIPI2: WD repeat domain, phosphoinositide interacting 2; XRN2: 5'-3' exoribonuclease 2.

Cash transfers and child nutritional outcomes: a systematic review and meta-analysis.

BACKGROUND: Cash transfer (CT) programmes are implemented widely to alleviate poverty and provide safety nets to vulnerable households with children. However, evidence on the effects of CTs on child health and nutrition outcomes has been mixed. We systematically reviewed evidence of the impact of CTs on child nutritional status and selected proximate determinants. METHODS: We searched articles published between January 1997 and September 2018 using Agris, Econlit, Eldis, IBSS, IDEAS, IFPRI, Google Scholar, PubMed and World Bank databases. We included studies using quantitative impact evaluation methods of CTs with sample sizes over 300, targeted to households with children under 5 years old conducted in countries with gross domestic product per capita below US$10 000 at baseline. We conducted meta-analysis using random-effects models to assess the impact of CT programmes on selected child nutrition outcomes and meta-regression analysis to examine the association of programme characteristics with effect sizes. RESULTS: Out of 2862 articles identified, 74 articles were eligible for inclusion. We find that CTs have significant effects of 0.03±0.03 on height-for-age z-scores (p<0.03) and a decrease of 2.1% in stunting (95% CI -3.5% to -0.7%); consumption of animal-source foods (4.5%, 95% CI 2.9% to 6.0%); dietary diversity (0.73, 95% CI 0.28 to 1.19) and diarrhoea incidence (-2.7%, 95% CI -5.4% to -0.0%; p<0.05). The effects of CTs on weight-for-age z-scores and wasting were not significant (0.02, 95% CI -0.03 to 0.08; p<0.42) and (1.2%, 95% CI: -0.1% to 2.5%; p<0.07), respectively. We found that specific programme characteristics differentially modified the effect on the nutrition outcomes studied. CONCLUSION: We found that CT programmes targeted to households with young children improved linear growth and contributed to reduced stunting. We found that the likely pathways were through increased dietary diversity, including through the increased consumption of animal-source foods and reduced incidence of diarrhoea. With heightened interest in nutrition-responsive social protection programmes to improve child nutrition, we make recommendations to inform the design and implementation of future programmes.

Widespread intron retention impairs protein homeostasis in C9orf72 ALS brains.

The GGGGCC hexanucleotide expansion in C9orf72 (C9) is the most frequent known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), yet a clear understanding of how C9 fits into the broader context of ALS/FTD pathology has remained lacking. The repetitive RNA derived from the C9 repeat is known to sequester hnRNPH, a splicing regulator, into insoluble aggregates, resulting in aberrant alternative splicing. Furthermore, hnRNPH insolubility and altered splicing of a robust set of targets have been observed to correlate in C9 and sporadic ALS/FTD patients alike, suggesting that changes along this axis are a core feature of disease pathogenesis. Here, we characterize previously uncategorized RNA splicing defects involving widespread intron retention affecting almost 2000 transcripts in C9ALS/FTD brains exhibiting a high amount of sequestered, insoluble hnRNPH. These intron retention events appear not to alter overall expression levels of the affected transcripts but rather the protein-coding regions. These retained introns affect transcripts in multiple cellular pathways predicted to be involved in C9 as well as sporadic ALS/FTD etiology, including the proteasomal and autophagy systems. The retained intron pre-mRNAs display a number of characteristics, including enrichment of hnRNPH-bound splicing enhancer motifs and a propensity for G-quadruplex (G-Q) formation, linking the defective splicing directly to high amounts of sequestered hnRNPH. Together, our results reveal previously undetected splicing defects in high insoluble hnRNPH-associated C9ALS brains, suggesting a feedback between effective RNA-binding protein dosage and protein quality control in C9, and perhaps all, ALS/FTD.

Widespread transcript shortening through alternative polyadenylation in secretory cell differentiation.

Most eukaryotic genes produce alternative polyadenylation (APA) isoforms. Here we report that, unlike previously characterized cell lineages, differentiation of syncytiotrophoblast (SCT), a cell type critical for hormone production and secretion during pregnancy, elicits widespread transcript shortening through APA in 3'UTRs and in introns. This global APA change is observed in multiple in vitro trophoblast differentiation models, and in single cells from placentas at different stages of pregnancy. Strikingly, the transcript shortening is unrelated to cell proliferation, a feature previously associated with APA control, but instead accompanies increased secretory functions. We show that 3'UTR shortening leads to transcripts with higher mRNA stability, which augments transcriptional activation, especially for genes involved in secretion. Moreover, this mechanism, named secretion-coupled APA (SCAP), is also executed in B cell differentiation to plasma cells. Together, our data indicate that SCAP tailors the transcriptome during formation of secretory cells, boosting their protein production and secretion capacity.

ALS/FTD-associated protein FUS induces mitochondrial dysfunction by preferentially sequestering respiratory chain complex mRNAs.

Dysregulation of the DNA/RNA-binding protein FUS causes certain subtypes of ALS/FTD by largely unknown mechanisms. Recent evidence has shown that FUS toxic gain of function due either to mutations or to increased expression can disrupt critical cellular processes, including mitochondrial functions. Here, we demonstrate that in human cells overexpressing wild-type FUS or expressing mutant derivatives, the protein associates with multiple mRNAs, and these are enriched in mRNAs encoding mitochondrial respiratory chain components. Notably, this sequestration leads to reduced levels of the encoded proteins, which is sufficient to bring about disorganized mitochondrial networks, reduced aerobic respiration and increased reactive oxygen species. We further show that mutant FUS associates with mitochondria and with mRNAs encoded by the mitochondrial genome. Importantly, similar results were also observed in fibroblasts derived from ALS patients with FUS mutations. Finally, we demonstrate that FUS loss of function does not underlie the observed mitochondrial dysfunction, and also provides a mechanism for the preferential sequestration of the respiratory chain complex mRNAs by FUS that does not involve sequence-specific binding. Together, our data reveal that respiratory chain complex mRNA sequestration underlies the mitochondrial defects characteristic of ALS/FTD and contributes to the FUS toxic gain of function linked to this disease spectrum.