De novo STXBP1 Mutations in Two Patients With Developmental Delay With or Without Epileptic Seizures

Objectives: Mutations in the STXBP1 gene have been associated with epileptic encephalopathy. Previous studies from in vitro neuroblastoma 2A cells showed that haploinsufficiency of STXBP1 is the mechanism for epileptic encephalopathy. In this ex vivo study, STXPB1 DNA mutations and RNA expression were assessed from two patients to help understand the impact of STXBP1 mutations on the disease etiology and mechanism.Methods: Microarray analysis and DNA sequencing were performed on two children with development delay, one with and one without infantile spasms. Different pathogenic mutations of STXBP1 were identified in the patients and RNA expression of STXPB1 was then performed by RT-Q-PCR on RNA extracted from blood samples of each patient.Results: Pathogenic deletion [of exons 13–20 and 3′ downstream of STXBP1] and nonsense mutation [c.1663G>T (p.Glu555X) in exon 18 of STXBP1] were detected from the two patients, respectively. RNA analysis showed that 1) the deletion mediated RNA decay, and that 2) no RNA decay was identified for the nonsense mutation at codon 555 which predicts a truncated STXBP1 protein.Significance: Our RNA expression analyses from the patient blood samples are the first ex vivo studies to support that both haploinsufficiency and truncation of STXBP1 protein (either dominant negative or haploinsufficiency) are causative mechanisms for epileptic encephalopathies, intellectual disability and developmental delay. The RNA assay also suggests that escape from nonsense-mediated RNA decay is possible when the nonsense mutation resides <50 nucleotides upstream of the last coding exon-exon junction even in the presence of additional non-coding exons that are 3′ downstream of the last coding exon.

Introns encode dsRNAs undetected by RIG-I/MDA5/interferons and sensed via RNase L

Double-stranded RNA (dsRNA), a hallmark viral material that activates antiviral interferon (IFN) responses, can appear in human cells also in the absence of viruses. We identify phosphorothioate DNAs (PS DNAs) as triggers of such endogenous dsRNA (endo-dsRNA). PS DNAs inhibit decay of nuclear RNAs and induce endo-dsRNA via accumulation of high levels of intronic and intergenic inverted retroelements (IIIR). IIIRs activate endo-dsRNA responses distinct from antiviral defense programs. IIIRs do not turn on transcriptional RIG-I/MDA5/IFN signaling, but they trigger the dsRNA-sensing pathways of OAS3/RNase L and PKR. Thus, nuclear RNA decay and nuclear-cytosolic RNA sorting actively protect from these innate immune responses to self. Our data suggest that the OAS3/RNase L and PKR arms of innate immunity diverge from antiviral IFN responses and monitor nuclear RNA decay by sensing cytosolic escape of IIIRs. OAS3 provides a receptor for IIIRs, whereas RNase L cleaves IIIR-carrying introns and intergenic RNAs.

Escherichia coli ribosomal protein S1 enhances the kinetics of ribosome biogenesis and RNA decay

Escherichia coli ribosomal protein S1 is essential for translation initiation of mRNAs and for cellular viability. Two oligonucleotide binding (OB)-fold domains located in the C-terminus of S1 are dispensable for growth, but their deletion causes a cold-shock phenotype, loss of motility and deregulation of RNA mediated stress responses. Surprisingly, the expression of the small regulatory RNA RyhB and one of its repressed target mRNA, sodB, are enhanced in the mutant strain lacking the two OB domains. Using in vivo and in vitro approaches, we show that RyhB retains its capacity to repress translation of target mRNAs in the mutant strain but becomes deficient in triggering rapid turnover of those transcripts. In addition, the mutant is defective in of the final step of the RNase E-dependent maturation of the 16S rRNA. This work unveils an unexpected function of S1 in facilitating ribosome biogenesis and RyhB-dependent mRNA decay mediated by the RNA degradosome. Through its RNA chaperone activity, S1 participates to the coupling between ribosome biogenesis, translation, and RNA decay.

Zinc finger protein ZFP36L1 inhibits flavivirus infection by both 5′-3′ XRN1 and 3′-5′ RNA-exosome RNA decay pathways.

Zinc-finger protein 36, CCCH type-like 1 (ZFP36L1), containing tandem CCCH-type zinc-finger motifs with an RNA-binding property, plays an important role in cellular RNA metabolism mainly via RNA decay pathways. Recently, we demonstrated that human ZFP36L1 has potent antiviral activity against influenza A virus infection. However, its role in the host defense response against flaviviruses has not been addressed. Here, we demonstrate that ZFP36L1 functions as a host innate defender against flaviviruses, including Japanese encephalitis virus (JEV) and dengue virus (DENV). Overexpression of ZFP36L1 reduced JEV and DENV infection, and ZFP36L1 knockdown enhanced viral replication. ZFP36L1 destabilized the JEV genome by targeting and degrading viral RNA mediated by both 5′-3′ XRN1 and 3′-5′ RNA-exosome RNA decay pathways. Mutation in both zinc-finger motifs of ZFP36L1 disrupted RNA-binding and antiviral activity. Furthermore, the viral RNA sequences specifically recognized by ZFP36L1 were mapped to the 3'-untranslated region of the JEV genome with the AU-rich element (AUUUA) motif. We extend the function of ZFP36L1 to host antiviral defense by directly binding and destabilizing the viral genome via recruiting cellular mRNA decay machineries. Importance Cellular RNA-binding proteins are among the first lines of defense against various viruses, particularly RNA viruses. ZFP36L1 belongs to the CCCH-type zinc-finger protein family and has RNA-binding activity; it has been reported to directly bind to the AU-rich elements (AREs) of a subset of cellular mRNAs and then lead to mRNA decay by recruiting mRNA degrading enzymes. However, the antiviral potential of ZFP36L1 against flaviviruses has not yet been fully demonstrated. Here, we reveal the antiviral potential of human ZFP36L1 against Japanese encephalitis virus (JEV) and dengue virus (DENV). ZFP36L1 specifically targeted the ARE motif within viral RNA and triggered the degradation of viral RNA transcripts via cellular degrading enzymes, 5′-3′ XRN1 and 3′-5′ RNA exosome. These findings provide mechanistic insights into how human ZFP36L1 serves as a host antiviral factor to restrict flavivirus replication.

RNA Decay Controls the Kinetics of the Angiotensin II Gene Expression Response

Complex cellular resonses require the temporal coordination of stimulus-induced gene expression programs. Angiotensin II (AngII) is the active 8 amino acid peptide in the renin-angiotensin-aldosterone system that controls blood pressure and fluid balance. AngII binds to type I angiotensin receptor in the adrenal cortex to initiate a cascade of temporally coordinated events leading to the production of aldosterone, a master regulator of blood pressure and volume. We stimulated a steroidogenic human cell line (H295R) with AngII and performed RNA-seq at twelve points. We identified twelve distict temporally distinct groups of gene expression responses each encoding functionally related proteins important for various steps of aldosterone productionl. Interstingly, the shape of the impulse response suggested a key role for RNA decay. Indeed, RNA decay rates in unstimulated H295R cells strongly correlated with the amplitude and peakiness of the gene expression response for each group of genes. We also found evidence for increases in RNA decay during the AngII response. Next, we selected candidate RBPs based on motif finding, adrenal specific expression, and AngII responsiveness. We performed an siRNA knockdown screen on these 22 candidates to identify RBPs that regulate aldosterone levels. Eight of these RBPs exhibited statistically significant changes in aldosterone for at least two independent siRNAs. Interestingly, multiple RBPs that promote RNA decay were found to suppress aldosterone production and induced in response to AngII-stimulation. These RBPs could be responsible for our observed increases in RNA decay. Altogether, these data support a model in which RNA decay is a critical regulator of the timing and strength of AngII-induced gene expression and ultimately aldosterone production.

Nonsense Mediated RNA Decay Is a Unique Vulnerability of Cancer Cells with SF3B1 and U2AF1 Mutations

Nonsense-mediated RNA decay (NMD) is well recognized as an RNA surveillance pathway that targets aberrant mRNAs with premature translation termination codons (PTCs) for degradation; however, its molecular mechanisms and roles in health and disease remain incompletely understood. In this study, we developed a novel reporter system that can accurately measure NMD activity in individual cells. By carrying out a genome-wide CRISPR/Cas9 knockout screen using this reporter system, we identified novel NMD-promoting factors, including multiple components of the SF3B complex and other U2 spliceosome factors. Interestingly, we also found that cells with mutations in the U2 spliceosome genes SF3B1 and U2AF1, which are commonly found in myelodysplastic syndrome (MDS) and cancers, have overall attenuated NMD activity. Furthermore, we found that compared to wild type cells, SF3B1 and U2AF1 mutant cells are more sensitive to NMD inhibition, a phenotype that is accompanied by elevated DNA replication obstruction, DNA damage and chromosomal instability. Remarkably, the sensitivity of spliceosome mutant cells to NMD inhibition could be rescued by overexpression of RNase H1, which removes R-loops in the genome. Together, our findings shed new light on the functional interplay between NMD and RNA splicing and suggest a novel strategy for the treatment of MDS and cancers with spliceosome mutations.