Helicase Domain Research Articles

Abstract A037: Investigating the role of the somatic DDX41 variant in the context of DDX41 hereditary myeloid malignancy syndromes (HMMS)

Abstract Heterozygous germline variants in DDX41 are identified in ~5% of patients with Myelodysplastic Syndrome (MDS) or Acute Myeloid Leukemia (AML). Recognition of the hereditary nature of such cancers and a better understanding of the factors that affect progression to malignancy has deep implications for health surveillance of patients and relatives that share a pathogenic DNA variant. DDX41 has a role in ribosome biogenesis and in opposing R-loop accumulation at promoters, but the mechanisms through which DDX41 loss drives cancer development are poorly understood. In a collaboration between Cambridge, UK and Mayo Clinic, USA we identified 85 patients with germline DDX41 variants [73/85 with germline pathogenic variants (GPV) and 12 with variants of uncertain significance (VUS)]. The majority were identified through targeted myeloid NGS upon presentation with AML (30/85) or MDS (44/85). Average age at diagnosis was 65 (35-93) with male predominance (57/85). The most common GPV was the NM_016222.4(DDX41):c.415_418dup; p.D140Gfs*2 (15/85). The majority (53.4% 39/73), also acquired a somatic variants in the other DDX41 allele (referred to as GPV+Som). The most common somatic variant (82% 32/39) was the R525H, a variant predicted to reduce the ATPase function of the helicase domain. We hypothesise that the markedly reduced DDX41 activity in cells with biallelic variants, is a key cause of progression to overt malignancy. In our cohort, we observed that somatic variants occurred at a similar frequency in association with start loss, truncating, splice, in-frame deletion, and missense GPV. However, whereas MDS was the predominant diagnosis within GPV (67.6% 23/34, AML 26.4% 9/34), AML was the most frequent diagnosis within GPV+Som (AML 51%.2 20/39, MDS 38.4% 15/39). Overall, there was a significantly higher likelihood of AML in the presence of a somatic variant (p<0.05), suggesting that there might be a dosage effect on the phenotype. This pattern was observed across all the GPV, with the exception of missense, suggesting that many might be benign polymorphysms. In addition, although somatic variants are typically subclonal (overall mean variant allele fraction VAF=9.87%, 3-27.8%), we observed a higher somatic VAF in AML over MDS cases (12.4% vs 7.7% respectively p<0.05). A lower bone marrow cellularity was also characteristic of GPV+Som vs GPV alone (mean observed/expected cellularity for age 0.9 vs 1.32 respectively p<0.05). In two cases, serial bone marrow morphology and NGS data were available, and we observed marked reduction in bone marrow cellularity upon acquisition of the R525H clone. The mechanisms of AML evolution appear different in DDX41 HMMS compared to sporadic cases. Interestingly, over half of our patients (21/34 GPV and 23/39 GPV+Som) did not exhibit additional somatic drivers. Our work suggests that acquisition of a somatic variant is a critical event in disease progression. Furthermore, the near universally subclonal nature of somatic variants suggest that they may affect disease phenotype through non-cell autonomous mechanisms. Citation Format: Ludovica Marando, Talha Badar, Yael Kusne, Audrey Morris, Terra Lasho, Abhishek Mangaonkar, Christy Finke, Carles Crawley, James M. Foran, Aref Al-Kali, Andrew King, Hassan Alkhateeb, Naseema Gangat, Chi Wong, Rong He, David Viswanatha, Faisal Basheer, Mark Litzow, Alejandro Ferrer, George Vassiliou, Mrinal Patnaik. Investigating the role of the somatic DDX41 variant in the context of DDX41 hereditary myeloid malignancy syndromes (HMMS) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A037.

The helicase domain of human Dicer prevents RNAi-independent activation of antiviral and inflammatory pathways

In mammalian somatic cells, the relative contribution of RNAi and the type I interferon response during viral infection is unclear. The apparent inefficiency of antiviral RNAi might be due to self-limiting properties and mitigating co-factors of the key enzyme Dicer. In particular, the helicase domain of human Dicer appears to be an important restriction factor of its activity. Here, we study the involvement of several helicase-truncated mutants of human Dicer in the antiviral response. All deletion mutants display a PKR-dependent antiviral phenotype against certain viruses, and one of them, Dicer N1, acts in a completely RNAi-independent manner. Transcriptomic analyses show that many genes from the interferon and inflammatory response pathways are upregulated in Dicer N1 expressing cells. We show that some of these genes are controlled by NF-kB and that blocking this pathway abrogates the antiviral phenotype of Dicer N1. Our findings highlight the crosstalk between Dicer, PKR, and the NF-kB pathway, and suggest that human Dicer may have repurposed its helicase domain to prevent basal activation of antiviral and inflammatory pathways.

Immunity to Tick-Borne Encephalitis Virus NS3 Protein Induced with a Recombinant Modified Vaccinia Virus Ankara Fails to Afford Mice Protection against TBEV Infection.

Tick-borne encephalitis (TBE) is a serious neurological disease caused by TBE virus (TBEV). Because antiviral treatment options are not available, vaccination is the key prophylactic measure against TBEV infections. Despite the availability of effective vaccines, cases of vaccination breakthrough infections have been reported. The multienzymatic non-structural protein 3 (NS3) of orthoflaviviruses plays an important role in polyprotein processing and virus replication. In the present study, we evaluated NS3 of TBEV as a potential vaccine target for the induction of protective immunity. To this end, a recombinant modified vaccinia virus Ankara that drives the expression of the TBEV NS3 gene (MVA-NS3) was constructed. MVA-NS3 was used to immunize C57BL/6 mice. It induced NS3-specific immune responses, in particular T cell responses, especially against the helicase domain of NS3. However, MVA-NS3-immunized mice were not protected from subsequent challenge infection with a lethal dose of the TBEV strain Neudoerfl, indicating that in contrast to immunity to prME and NS1, NS3-specific immunity is not an independent correlate of protection against TBEV in this mouse model.

Cryo-EM structures of human DICER dicing a pre-miRNA substrate.

Dicer, a multi-domain ribonuclease III (RNase III) protein, is crucial for gene regulation via RNA interference. It processes hairpin-like precursors into microRNAs (miRNAs) and long double-stranded RNAs (dsRNAs) into small interfering RNAs (siRNAs). During the "dicing" process, the miRNA or siRNA substrate is stably anchored and cleaved by Dicer's RNase III domain. Although numerous studies have investigated long dsRNA cleavage by Dicer, the specific mechanism by which human Dicer (hDICER) processes pre-miRNA remains unelucidated. This review introduces the recently revealed hDICER structure bound to pre-miRNA uncovered through cryo-electron microscopy and compares it with previous reports describing Dicer. The domain-wise movements of the helicase and dsRNA-binding domain (dsRBD) and specific residues involved in substrate sequence recognition have been identified. During RNA substrate binding, the hDICER apical domains and dsRBD recognize the pre-miRNA termini and cleavage site, respectively. Residue rearrangements in positively charged pockets within the apical domain influence substrate recognition and cleavage site determination. The specific interactions between dsRBD positively charged residues and nucleotide bases near the cleavage site emphasize the significance of cis-acting elements in the hDICER processing mechanism. These findings provide valuable insights for understanding hDICER-related diseases.

Structural insights into branch site proofreading by human spliceosome.

Selection of the pre-mRNA branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is crucial to prespliceosome (A complex) assembly. The RNA helicase PRP5 proofreads BS selection but the underlying mechanism remains unclear. Here we report the atomic structures of two sequential complexes leading to prespliceosome assembly: human 17S U2 snRNP and a cross-exon pre-A complex. PRP5 is anchored on 17S U2 snRNP mainly through occupation of the RNA path of SF3B1 by an acidic loop of PRP5; the helicase domain of PRP5 associates with U2 snRNA; the BS-interacting stem-loop (BSL) of U2 snRNA is shielded by TAT-SF1, unable to engage the BS. In the pre-A complex, an initial U2-BS duplex is formed; the translocated helicase domain of PRP5 stays with U2 snRNA and the acidic loop still occupies the RNA path. The pre-A conformation is specifically stabilized by the splicing factors SF1, DNAJC8 and SF3A2. Cancer-derived mutations in SF3B1 damage its association with PRP5, compromising BS proofreading. Together, these findings reveal key insights into prespliceosome assembly and BS selection or proofreading by PRP5.

Structural insight into the assembly and working mechanism of helicase-primase D5 from Mpox virus.

The Mpox pandemic, caused by the Mpox virus (or monkeypox virus, MPXV), has gained global attention. The D5 protein, a putative helicase-primase found in MPXV, plays a vital role in viral replication and genome uncoating. Here we determined multiple cryo-EM structures of full-length hexameric D5 in diverse states. These states were captured during ATP hydrolysis while moving along the single-stranded DNA (ssDNA) track. Through comprehensive structural analysis combined with the helicase activity system, we revealed that when the primase domain is truncated or the interaction between the primase and helicase domains is disrupted, the double-stranded DNA (dsDNA) unwinds into ssDNA, suggesting a critical regulatory role of the primase domain. Two transition states bound with ssDNA substrate during unwinding reveals that two ATP molecules were consumed to drive DNA moving forward two nucleotides. Collectively, our findings shed light on the molecular mechanism that links ATP hydrolysis to the DNA unwinding in poxviruses.

YLR419W is the homolog of the mammalian translation initiation factor DHX29.

27 years after the yeast genome sequencing, the function of many ORFs remain unknown. Despite the evolutionary distance between human and yeast, homology with the conserved DEAH/DExH-box helicase domains allowed us to list DHX29, DHX36 and DHX57 as three putative homologs of the yeast Ylr419wp. Functional studies first linked the Ylr419w protein to the translating ribosome and cross-linking and analysis of cDNA (CRAC) experiments determined the precise region of Ylr419wp in contact with the ribosome. It corresponds to the loop of the h16 helix in the 18S rRNA designing the translation initiation factor DHX29, as the functional homolog of Ylr419wp.

Harmonin homology domain-mediated interaction of RTEL1 helicase with RPA and DNA provides insights into its recruitment to DNA repair sites.

The regulator of telomere elongation helicase 1 (RTEL1) playsrolesin telomere DNA maintenance, DNA repair, and genome stability by dismantling D-loops and unwinding G-quadruplex structures. RTEL1 comprises a helicase domain, two tandem harmonin homology domains 1&2 (HHD1 and HHD2), and a Zn2+-binding RING domain. In vitro D-loop disassembly by RTEL1 is enhanced in the presence of replication protein A (RPA). However, the mechanism of RTEL1 recruitment at non-telomeric D-loops remainsunknown. In this study, we have unravelled a direct physical interaction between RTEL1 and RPA. Under DNA damage conditions, we showed that RTEL1 and RPA colocalise in the cell. Coimmunoprecipitation showed that RTEL1 and RPA interact, and the deletion of HHDs of RTEL1 significantly reduced this interaction. NMR chemical shift perturbations (CSPs) showed that RPA uses its 32C domain to interact with the HHD2 of RTEL1.Interestingly, HHD2 also interacted with DNA in thein vitro experiments. HHD2 structure was determined using X-ray crystallography, and NMR CSPs mapping revealed that both RPA32C and DNAcompetitivelybind toHHD2 on an overlapping surface. These results establish novel roles of accessory HHDs in RTEL1's functionsand provide mechanistic insights into the RPA-mediated recruitment of RTEL1to DNA repairsites.

Human RTEL1 Interacts with KPNB1 (Importin β) and NUP153 and Connects Nuclear Import to Nuclear Envelope Stability in S-Phase.

Regulator of TElomere Length Helicase 1 (RTEL1) is a helicase required for telomere maintenance and genome replication and repair. RTEL1 has been previously shown to participate in the nuclear export of small nuclear RNAs. Here we show that RTEL1 deficiency leads to a nuclear envelope destabilization exclusively in cells entering S-phase and in direct connection to origin firing. We discovered that inhibiting protein import also leads to similar, albeit non-cell cycle-related, nuclear envelope disruptions. Remarkably, overexpression of wild-type RTEL1, or of its C-terminal part lacking the helicase domain, protects cells against nuclear envelope anomalies mediated by protein import inhibition. We identified distinct domains in the C-terminus of RTEL1 essential for the interaction with KPNB1 (importin β) and NUP153, respectively, and we demonstrated that, on its own, the latter domain can promote the dynamic nuclear internalization of peptides that freely diffuse through the nuclear pore. Consistent with putative functions exerted in protein import, RTEL1 can be visualized on both sides of the nuclear pore using high-resolution microscopy. In all, our work points to an unanticipated, helicase-independent, role of RTEL1 in connecting both nucleocytoplasmic trafficking and nuclear envelope integrity to genome replication initiation in S-phase.

Abstract PR007: Discovery of HRO761, a novel, first-in-class clinical stage WRN inhibitor with potent and selective anti-tumor activity in cancers with microsatellite instability

Abstract The RecQ DNA helicase WRN was identified as a synthetic lethal target in tumors with microsatellite instability (MSI) by several genetic screens. Despite recent advances in the treatment of MSI tumors by immune checkpoint inhibitors, a significant proportion of patients still fails to respond to or relapses after single agent anti-PD1 or combination of anti-PD1 plus anti-CTLA4 treatments. We present the biochemical, cellular and pharmacological characterization of the first potent and selective WRN helicase inhibitor, HRO761. We show that HRO761 is an allosteric WRN inhibitor that binds at the interface of the D1 and D2 helicase domains, locking WRN in an inactive conformation. Pharmacological inhibition of WRN by HRO761 recapitulates the phenotype observed by WRN genetic suppression, leading to activation of the DNA damage response and inhibition of tumor cell growth selectively in MSI but not in MSS cells. We further show that WRN inhibition leads to WRN protein degradation only in MSI tumor cells. Although HRO761 activates a p53-related response, anti-proliferative effects were observed in both p53 wild-type as well as p53 null or mutated tumor cells. HRO761 exposure in mice is linear with oral dose and results in dose-dependent in vivo DNA damage induction and tumor growth inhibition in MSI cell- and patient-derived xenograft models. Altogether these findings represent preclinical pharmacological validation of WRN as a selective therapeutic target in MSI cancers. A global first in human clinical trial with HRO761 (NCT05838768) is currently ongoing to assess the safety, tolerability and preliminary anti-tumor activity in patients with MSI colorectal cancer and other MSI solid tumors. Citation Format: Marta Cortes-Cros, Henrik Moebitz, Stephanie Barbe, Jutta Blank, Vincent Bordas, Giorgia Clementi, Ernesta Dammassa, Andrea Decker, Noemi Di Nanni, Ruben de Kanter, Marion Dourdoigne, Elena Gavioli, Stephane Ferretti, Marc Hattenberger, Christelle Hemmerlin, Jacques Hamon, Juergen Hinrichs, Isabel Jaco, Dragana Jankovic, Eloisa Jimenez Nunez, Yifang Li, Hans-Joerg Martus, Michele Moschetta, Cornelia Quadt, Markus Reschke, Vincent Romanet, Clemens Scheufler, Joseph Schoepfer, Ross Strang, Maxime Therier, Hans Voshol, Markus Wartmann, Sarah Welly, Monica Pham, Laurent Laborde. Discovery of HRO761, a novel, first-in-class clinical stage WRN inhibitor with potent and selective anti-tumor activity in cancers with microsatellite instability [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR007.

Pathogenic Mechanisms of DDX41 Mutations in the Development of Myeloid Malignancies

DDX41 is a newly identified leukemia predisposition gene encoding an RNA helicase, whose germline mutations are tightly associated with late-onset myeloid malignancies. Importantly, germline DDX41 mutations were also found in as many as ~7 % of sporadic cases of high-risk MDS, conferring the largest germline risk for myeloid malignancies. In typical cases, a germline loss-of-function allele (most commonly p.A500fs or p.D140fs, depending on the ethnicity) is compounded by a somatic missense mutation affecting the helicase domain in the remaining allele (p.R525H). However, the molecular mechanism by which DDX41 mutations lead to myeloid neoplasms have not fully been elucidated. To clarify the role of these distinct DDX41 alleles, we generated mice models carrying either or both of conditional/constitutive Ddx41 knock-out (KO) and conditional R525H knock-in (KI) alleles. Vav1-Cre mediated homozygous deletion of Ddx41 resulted in embryonic lethality, suggesting that Ddx41 is indispensable for normal hematopoiesis. Next, by crossing these mice and further breeding with Rosa26-CreERT2 transgenic mice, we engineered mice that were wild-type for Ddx41 ( Ddx41 +/+), heterozygous Ddx41 KO( Ddx41 +/-), homozygous Ddx41 KO( Ddx41 -/-), heterozygous for the Ddx41 R525H mutation ( Ddx41 R525H/+), or hemizygous for the Ddx41 R525H mutation( Ddx41 R525H/-), in which expression of the mutant allele was induced by tamoxifen administration. First, we assessed cell intrinsic effects of these Ddx41 alleles, using noncompetitive transplantation experiments. Shortly after tamoxifen administration, most of the recipient mice that were transplanted with BM from Ddx41 -/- or Ddx41 R525H/- mice died within a month after CreERT2 induction due to severe BM failure (BMF), which was not observed in mice transplanted with BM from Ddx41 +/+, Ddx41 +/- or Ddx41 R525H/+ mice. By contrast, the mice transplanted with Ddx41 +/- or Ddx41 R525H/+ BM showed significantly reduced WBC counts and anemia in long-term observation in both primary and serial transplantations. Some of the Ddx41 +/- or Ddx41 R525H/+ BM-transplanted mice exhibited MDS-like phenotypes, showing ineffective hematopoiesis with evidence of erythroid dysplasia. Transcriptome analysis revealed that stem cells (Kit +Sca-1 -Lin low cells) derived from Ddx41 R525H/- BM-transplanted mice exhibited a significant upregulation of genes involved in innate immunity, including interferon stimulated genes, compared with stem cells derived from Ddx41 +/+ BM-transplanted mice. In addition, snoRNA and ribosomal genes were significantly deregulated in stem cells from Ddx41 -/- and Ddx41 R525H/- BM-transplanted mice, which could result in abnormal ribosome biogenesis and protein synthesis in Ddx41 mutant cells. We also assessed the reconstitution capacity of whole BM cells from different Ddx41 mutant mice in competitive transplantation experiments. The donor chimerism of Ddx41 -/- and Ddx41 R525H/- BM-transplanted mice in PB was markedly reduced compared to Ddx41 +/+ BM-transplanted mice. In contrast, Ddx41 +/- and Ddx41 R525H/+ BM-transplanted micedid not show significant changes in competitive bone marrow reconstitution compared to Ddx41 +/+ BM-transplanted mice. Given that the MDS clones bearing DDX41 R525H somatic alleles are typically observed as a small subclone in patients, we next co-transplanted Ddx41 +/+- and Ddx41 525H/--derived BM cells with Ddx41 +/+- or Ddx41 +/--derived BM cells at the ratio of 1:9. Recipient mice showed significantly reduced WBC counts when Ddx41 +/+- or Ddx41 +/- were co-transplanted with Ddx41 525H/- -derived BM, suggesting that Ddx41 525H/--derived hematopoietic cells have negative effect on normal hematopoiesis. In summary, compound biallelic loss-of function and R525 alleles led to severe BM failure. Monoallelic Ddx41 loss-of function and R525H knock-in alleles, by contrast, are compatible with hematopoiesis but associated with an impairment of hematopoiesis and the development of MDS with ageing, where activated innate immunity, impaired RNA metabolism and ribosome functions may play important roles.

A tug-of-war to control plant emission of an airborne alarm signal

Aphids represent a major threat to crops. Hundreds of different viruses are aphid-borne. Upon aphid attack, plants release volatile organic compounds (VOCs) as airborne alarm signals to turn on the airborne defense (AD) of neighboring plants, thereby repelling aphids as well as reducing aphid fitness and virus transmission. This phenomenon provides a critical community-wide plant protection to fend off aphids, but the underlying molecular basis remains undetermined for a long time. In a recent article, Gong et al. established the NAC2-SAMT1 module as the core component regulating the emission of methyl-salicylate (MeSA), a major component of VOCs in aphid-attacked plants. Furthermore, they showed that SABP2 protein is critical for the perception of volatile MeSA signal by converting MeSA to Salicylic Acid (SA), which is the cue to elicit AD against aphids at the community level. Moreover, they showed that multiple viruses use a conserved glycine residue in the ATP-dependent helicase domain in viral proteins to shuttle NAC2 from the nucleus to the cytoplasm for degradation, leading to the attenuation of MeSA emission and AD. These findings illuminate the functional roles of key regulators in the complex MeSA-mediated airborne defense process and a counter-defense mechanism used by viruses, which has profound significance in advancing the knowledge of plant-pathogen interactions as well as providing potential targets for gene editing-based crop breeding.

Black carp LGP2 suppresses RIG-I mediated IFN signaling during the antiviral innate immunity

Laboratory of genetics and physiology 2 (LGP2), a member of retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), has been reported to play different roles in IFN signaling in both mammals and teleost fish. In our previous study, black carp (Mylopharyngodon piceus) LGP2 (bcLGP2) has been characterized to positively regulate melanoma differentiation-associated gene 5 (MDA5). In this study, knockdown of bcLGP2 decreased the expression of host genes, including bcIFNb, bcPKR, bcMx1, and bcViperin, and also attenuated the antiviral capability of host cells. The relationship between bcLGP2 and black carp RIG-Ib (bcRIG-Ib) has been explored. Dual-luciferase reporter assay and qRT-PCR assay indicated that bcLGP2 dampened bcRIG-Ib induced transcription of type I interferons (IFNs) and interferon-stimulated genes (ISGs), including PKR, ISG15, and Viperin. Consistently, the plaque assay identified that bcLGP2 attenuated bcRIG-Ib mediated antiviral ability against spring viremia of carp virus (SVCV). Co-immunoprecipitation assay identified the interaction between bcLGP2 and bcRIG-Ib, as well as bcLGP2 and bcRIG-Ib-CARD. And bcRIG-Ib-CARD mediated antiviral ability was also attenuated by bcLGP2. Truncation mutation analysis showed DExD/H-box Helicase domain of bcLGP2 possessed a similar inhibitory effect on bcRIG-Ib to that of bcLGP2, while the C-terminus repressor domain (CTD) presented little impact on bcRIG-Ib. Furthermore, bcLGP2 enhanced the K48-linked ubiquitination of bcRIG-Ib, promoting proteasome-dependent degradation of bcRIG-Ib. Thus, our data supported the conclusion that bcLGP2 interacted with and induced degradation of bcRIG-Ib through proteasome, leading to the dampened antiviral signaling mediated by bcRIG-Ib.

Inhibition of HSP90 distinctively modulates the global phosphoproteome of Leishmania mexicana developmental stages.

In the unicellular parasites Leishmania spp., the etiological agents of leishmaniasis, a complex infectious disease that affects 98 countries in 5 continents, chemical inhibition of HSP90 protein leads to differentiation from promastigote to amastigote stage. Recent studies indicate potential role for protein phosphorylation in the life cycle control of Leishmania. Also, recent studies suggest a fundamentally important role of RNA-binding proteins (RBPs) in regulating the downstream effects of the HSP90 inhibition in Leishmania. Phosphorylation-dephosphorylation dynamics of RBPs in higher eukaryotes serves as an important on/off switch to regulate RNA processing and decay in response to extracellular signals and cell cycle check points. In the current study, using a combination of highly sensitive TMT labeling-based quantitative proteomic MS and robust phosphoproteome enrichment, we show for the first time that HSP90 inhibition distinctively modulates global protein phosphorylation landscapes in the different life cycle stages of Leishmania, shedding light into a crucial role of the posttranslational modification in the differentiation of the parasite under HSP90 inhibition stress. We measured changes in phosphorylation of many RBPs and signaling proteins including protein kinases upon HSP90 inhibition in the therapeutically relevant amastigote stage. This work provides insights into the importance of HSP90-mediated protein cross-talks and regulation of phosphorylation in Leishmania, thus significantly expanding our knowledge of the posttranslational modification in Leishmania biology.

Phage single-stranded DNA-binding protein or host DNA damage triggers the activation of the AbpAB phage defense system.

Although numerous phage defense systems have recently been discovered in bacteria, how these systems defend against phage propagation or sense phage infections remains unclear. The Escherichia coli AbpAB defense system targets several lytic and lysogenic phages harboring DNA genomes. A phage-encoded single-stranded DNA-binding protein, Gp32, activates this system similar to other phage defense systems such as Retron-Eco8, Hachiman, ShosTA, Nhi, and Hna. DNA replication inhibitors or defects in DNA repair factors activate the AbpAB system, even without phage infection. This is one of the few examples of activating phage defense systems without phage infection or proteins. The AbpAB defense system may be activated by sensing specific DNA-protein complexes.

Schlafen-5 inhibits LINE-1 retrotransposition

Long interspersed element 1 (LINE-1) is the only currently known active autonomous transposon in humans, and its retrotransposition may cause deleterious effects on the structure and function of host cell genomes and result in sporadic genetic diseases. Host cells therefore developed defense strategies to restrict LINE-1 mobilization. In this study, we demonstrated that IFN-inducible Schlafen5 (SLFN5) inhibits LINE-1 retrotransposition. Mechanistic studies revealed that SLFN5 interrupts LINE-1 ribonucleoprotein particle (RNP) formation, thus diminishing nuclear entry of the LINE-1 RNA template and subsequent LINE-1 cDNA production. The ability of SLFN5 to bind to LINE-1 RNA and the involvement of the helicase domain of SLFN5 in its inhibitory activity suggest a mechanism that SLFN5 binds to LINE-1 RNA followed by dissociation of ORF1p through its helicase activity, resulting in impaired RNP formation. These data highlight a new mechanism of host cells to restrict LINE-1 mobilization.

Complete genome sequence of a novel fusarivirus from the phytopathogenic fungus Fusarium sp.

Fusarium diseases include wilts, blights, rots, and cankers of many horticultural, field, ornamental, and forest crops in both agricultural and natural ecosystems, and they significantly hinder food plant production. Here, we describe a novel mycovirus, tentatively designated as "Fusarium fusarivirus 1" (FuFV1), which was discovered in an isolate of the phytopathogenic fungus Fusarium sp. FuFV1 has a positive-sense single-stranded RNA (+ssRNA) genome of 6,391 nucleotides (nt) containing three open reading frames (ORFs). ORF1 encodes a large polypeptide of 1,501 amino acids (aa) with conserved RNA-dependent RNA polymerase (RdRp) and helicase (Hel) domains. ORF2, overlapping ORF1 by 122 nucleotides, encodes a polypeptide with a conserved Smc domain. The third and smaller ORF (ORF3) encodes a polypeptide with an unknown function. BLASTp analysis of the ORF1-encoded polypeptide revealed that FuFV1 shares the highest aa sequence similarity (68.5% identity, E-value 0.0) with Fusarium poae fusarivirus 1 (FpFV1, genus Alphafusarivirus). Phylogenetic analysis of the RdRp and helicase (Hel) sequences indicated that FuFV1 clustered closely with FpFV1 in a separate branch within the clade containing members of the genus Alphafusarivirus. Based on these results, we propose that FuFV1 should be considered a novel mycovirus belonging to the genus Alphafusarivirus of the family Fusariviridae.

Chromatin remodeling of histone H3 variants by DDM1 underlies epigenetic inheritance of DNA methylation

Nucleosomes block access to DNA methyltransferase, unless they are remodeled by DECREASE in DNA METHYLATION 1 (DDM1LSH/HELLS), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 promotes replacement of histone variant H3.3 by H3.1. In ddm1 mutants, DNA methylation is partly restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2Å of DDM1 with a variant nucleosome reveals engagement with histone H3.3 near residues required for assembly and with the unmodified H4 tail. An N-terminal autoinhibitory domain inhibits activity, while a disulfide bond in the helicase domain supports activity. DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1Dnmt1, but is blocked by H4K16 acetylation. The male germline H3.3 variant MGH3/HTR10 is resistant to remodeling by DDM1 and acts as a placeholder nucleosome in sperm cells for epigenetic inheritance.

Structural mechanism of R2D2 and Loqs-PD synergistic modulation on DmDcr-2 oligomers

Small interference RNAs are the key components of RNA interference, a conserved RNA silencing or viral defense mechanism in many eukaryotes. In Drosophila melanogaster, Dicer-2 (DmDcr-2)-mediated RNAi pathway plays important roles in defending against viral infections and protecting genome integrity. During the maturation of siRNAs, two cofactors can regulate DmDcr-2’s functions: Loqs-PD that is required for dsRNA processing, and R2D2 that is essential for the subsequent loading of siRNAs into effector Ago2 to form RISC complexes. However, due to the lack of structural information, it is still unclear whether R2D2 and Loqs-PD affect the functions of DmDcr-2 simultaneously. Here we present several cryo-EM structures of DmDcr-2/R2D2/Loqs-PD complex bound to dsRNAs with various lengths by the Helicase domain. These structures revealed that R2D2 and Loqs-PD can bind to different regions of DmDcr-2 without interfering with each other. Furthermore, the cryo-EM results demonstrate that these complexes can form large oligomers and assemble into fibers. The formation and depolymerization of these oligomers are associated with ATP hydrolysis. These findings provide insights into the structural mechanism of DmDcr-2 and its cofactors during siRNA processing.

HELZ2: a new, interferon-regulated, human 3'-5' exoribonuclease of the RNB family is expressed from a non-canonical initiation codon.

Proteins containing a RNB domain, originally identified in Escherichia coli RNase II, are widely present throughout the tree of life. Many RNB proteins have 3'-5' exoribonucleolytic activity but some have lost catalytic activity during evolution. Database searches identified a new RNB domain-containing protein in human: HELZ2. Analysis of genomic and expression data combined with evolutionary information suggested that the human HELZ2 protein is produced from an unforeseen non-canonical initiation codon in Hominidae. This unusual property was confirmed experimentally, extending the human protein by 247 residues. Human HELZ2 was further shown to be an active ribonuclease despite the substitution of a key residue in its catalytic center. HELZ2 RNase activity is lost in cells from some cancer patients as a result of somatic mutations. HELZ2 harbors also two RNA helicase domains and several zinc fingers and its expression is induced by interferon treatment. We demonstrate that HELZ2 is able to degrade structured RNAs through the coordinated ATP-dependent displacement of duplex RNA mediated by its RNA helicase domains and its 3'-5' ribonucleolytic action. The expression characteristics and biochemical properties of HELZ2 support a role for this factor in response to viruses and/or mobile elements.