Unveiling the DHX15-G-patch interplay in retroviral RNA packaging.

We explored how a simple retrovirus, Mason-Pfizer monkey virus (M-PMV) to facilitate its replication process, utilizes DHX15, a cellular RNA helicase, typically engaged in RNA processing. Through advanced genetic engineering techniques, we showed that M-PMV recruits DHX15 by mimicking cellular mechanisms, relocating it from the nucleus to the cytoplasm to aid in viral assembly. This interaction is essential for the correct packaging of the viral genome and critical for its infectivity. Our findings offer unique insights into the mechanisms of viral manipulation of host cellular processes, highlighting a sophisticated strategy that viruses employ to leverage cellular machinery for their replication. This study adds valuable knowledge to the understanding of viral-host interactions but also suggests a common evolutionary history between cellular processes and viral mechanisms. This finding opens a unique perspective on the export mechanism of intron-retaining mRNAs in the packaging of viral genetic information and potentially develop ways to stop it.

A homozygous missense variant in YTHDC2 induces azoospermia in two siblings.

Male infertility is a complex multifactorial reproductive disorder with highly heterogeneous phenotypic presentations. Azoospermia is a medically non-manageable cause of male infertility affecting ∼1% of men. Precise etiology of azoospermia is not known in approximately three-fourth of the cases. To explore the genetic basis of azoospermia, we performed whole exome sequencing in two non-obstructive azoospermia affected siblings from a consanguineous Pakistani family. Bioinformatic filtering and segregation analysis of whole exome sequencing data resulted in the identification of a rare homozygous missense variant (c.962G>C, p. Arg321Thr) in YTHDC2, segregating with disease in the family. Structural analysis of the missense variant identified in our study and two previously reported functionally characterized missense changes (p. Glu332Gln and p. His327Arg) in mice showed that all these three variants may affect Mg2+ binding ability and helicase activity of YTHDC2. Collectively, our genetic analyses and experimental observations revealed that missense variant of YTHDC2 can induce azoospermia in humans. These findings indicate the important role of YTHDC2 deficiency for azoospermia and will provide important guidance for genetic counseling of male infertility.

RNA helicase SKIV2L limits antiviral defense and autoinflammation elicited by the OAS-RNase L pathway.

The OAS-RNase L pathway is one of the oldest innate RNA sensing pathways that leads to interferon (IFN) signaling and cell death. OAS recognizes viral RNA and then activates RNase L, which subsequently cleaves both cellular and viral RNA, creating "processed RNA" as an endogenous ligand that further triggers RIG-I-like receptor signaling. However, the IFN response and antiviral activity of the OAS-RNase L pathway are weak compared to other RNA-sensing pathways. Here, we discover that the SKIV2L RNA exosome limits the antiviral capacity of the OAS-RNase L pathway. SKIV2L-deficient cells exhibit remarkably increased interferon responses to RNase L-processed RNA, resulting in heightened antiviral activity. The helicase activity of SKIV2L is indispensable for this function, acting downstream of RNase L. SKIV2L depletion increases the antiviral capacity of OAS-RNase L against RNA virus infection. Furthermore, SKIV2L loss exacerbates autoinflammation caused by human OAS1 gain-of-function mutations. Taken together, our results identify SKIV2L as a critical barrier to OAS-RNase L-mediated antiviral immunity that could be therapeutically targeted to enhance the activity of a basic antiviral pathway.

Membrane Retention of West Nile Virus NS5 Depends on NS1 or NS3 for Enzymatic Activity.

West Nile virus (WNV) nonstructural protein 5 (NS5) possesses multiple enzymatic domains essential for viral RNA replication. During infection, NS5 predominantly localizes to unique replication organelles (ROs) at the rough endoplasmic reticulum (RER), known as vesicle packets (VPs) and convoluted membranes (CMs), with a portion of NS5 accumulating in the nucleus. NS5 is a soluble protein that must be in the VP, where its enzymatic activities are required for viral RNA synthesis. However, the mechanistic processes behind the recruitment of NS5 from the cytoplasm to the RER membrane remain unclear. Here, we utilize high-resolution confocal microscopy and sucrose density gradient ultracentrifugation to investigate whether the association of NS5 with other NS proteins contributes to its membrane recruitment and retention. We demonstrate that NS1 or NS3 partially influences the NS5 association with the membrane. We further demonstrate that processed NS5 is predominantly in the cytoplasm and nucleus, indicating that the processing of NS5 from the viral polyprotein does not contribute to its membrane localization. These observations suggest that other host or viral factors, such as the enwrapment of NS5 by the RO, may also be necessary for the complete membrane retention of NS5. Therefore, studies on the inhibitors that disrupt the membrane localization of WNV NS5 are warranted for antiviral drug development.

Stress granule formation helps to mitigate neurodegeneration.

Cellular stress pathways that inhibit translation initiation lead to transient formation of cytoplasmic RNA/protein complexes known as stress granules. Many of the proteins found within stress granules and the dynamics of stress granule formation and dissolution are implicated in neurodegenerative disease. Whether stress granule formation is protective or harmful in neurodegenerative conditions is not known. To address this, we took advantage of the alphavirus protein nsP3, which selectively binds dimers of the central stress granule nucleator protein G3BP and markedly reduces stress granule formation without directly impacting the protein translational inhibitory pathways that trigger stress granule formation. In Drosophila and rodent neurons, reducing stress granule formation with nsP3 had modest impacts on lifespan even in the setting of serial stress pathway induction. In contrast, reducing stress granule formation in models of ataxia, amyotrophic lateral sclerosis and frontotemporal dementia largely exacerbated disease phenotypes. These data support a model whereby stress granules mitigate, rather than promote, neurodegenerative cascades.

The divergent effects of G3BP orthologs on human stress granule assembly imply a centric role for the core protein interaction network.

Liquid-liquid phase separation (LLPS) mediated by G3BP1/2 proteins and non-translating mRNAs mediates stress granule (SG) assembly. We investigated the phylogenetic evolution of G3BP orthologs from unicellular yeast to mammals and identified both conserved and divergent features. The modular domain organization of G3BP orthologs is generally conserved. However, invertebrate orthologs displayed reduced capacity for SG assembly in human cells compared to vertebrate orthologs. We demonstrated that the protein-interaction network facilitated by the NTF2L domain is a crucial determinant of this specificity. The evolution of the G3BP1 network coincided with its exploitation by certain viruses, as evident from the interaction between viral proteins and G3BP orthologs in insects and vertebrates. We revealed the importance and divergence of the G3BP interaction network in human SG formation. Leveraging this network, we established a 7-component in vitro SG reconstitution system for quantitative studies. These findings highlight the significance of G3BP network divergence in the evolution of biological processes.

Senataxin RNA/DNA helicase promotes replication restart at co-transcriptional R-loops to prevent MUS81-dependent fork degradation.

Replication forks stalled at co-transcriptional R-loops can be restarted by a mechanism involving fork cleavage-religation cycles mediated by MUS81 endonuclease and DNA ligase IV (LIG4), which presumably relieve the topological barrier generated by the transcription-replication conflict (TRC) and facilitate ELL-dependent reactivation of transcription. Here, we report that the restart of R-loop-stalled replication forks via the MUS81-LIG4-ELL pathway requires senataxin (SETX), a helicase that can unwind RNA:DNA hybrids. We found that SETX promotes replication fork progression by preventing R-loop accumulation during S-phase. Interestingly, loss of SETX helicase activity leads to nascent DNA degradation upon induction of R-loop-mediated fork stalling by hydroxyurea. This fork degradation phenotype is independent of replication fork reversal and results from DNA2-mediated resection of MUS81-cleaved replication forks that accumulate due to defective replication restart. Finally, we demonstrate that SETX acts in a common pathway with the DEAD-box helicase DDX17 to suppress R-loop-mediated replication stress in human cells. A possible cooperation between these RNA/DNA helicases in R-loop unwinding at TRC sites is discussed.

R-loops and impaired autophagy trigger cGAS-dependent inflammation via micronuclei formation in Senataxin-deficient cells.

Senataxin is an evolutionarily conserved DNA/RNA helicase, whose dysfunctions are linked to neurodegeneration and cancer. A main activity of this protein is the removal of R-loops, which are nucleic acid structures capable to promote DNA damage and replication stress. Here we found that Senataxin deficiency causes the release of damaged DNA into extranuclear bodies, called micronuclei, triggering the massive recruitment of cGAS, the apical sensor of the innate immunity pathway, and the downstream stimulation of interferon genes. Such cGAS-positive micronuclei are characterized by defective membrane envelope and are particularly abundant in cycling cells lacking Senataxin, but not after exposure to a DNA breaking agent or in absence of the tumor suppressor BRCA1 protein, a partner of Senataxin in R-loop removal. Micronuclei with a discontinuous membrane are normally cleared by autophagy, a process that we show is impaired in Senataxin-deficient cells. The formation of Senataxin-dependent inflamed micronuclei is promoted by the persistence of nuclear R-loops stimulated by the DSIF transcription elongation complex and the engagement of EXO1 nuclease activity on nuclear DNA. Coherently, high levels of EXO1 result in poor prognosis in a subset of tumors lacking Senataxin expression. Hence, R-loop homeostasis impairment, together with autophagy failure and unscheduled EXO1 activity, elicits innate immune response through micronuclei formation in cells lacking Senataxin.

tRF-27 competitively Binds to G3BPs and Activates MTORC1 to Enhance HER2 Positive Breast Cancer Trastuzumab Tolerance.

About 20% of breast cancer patients are positive for HER2. The efficacy of current treatments is limited by primary and secondary resistance to trastuzumab. tRNA-derived fragments (tRFs) have shown crucial regulatory roles in various cancers. This study aimed to evaluate the role of tRF-27 in regulating the resistance of HER2-positive breast cancer against trastuzumab. tRF-27 was highly expressed in trastuzumab-resistant cells, and its expression level could predict the resistance to trastuzumab. High expression of tRF-27 promoted the growth and proliferation of trastuzumab-exposed cells. RNA-pulldown assay and mass spectrometry were performed to identify Ras GTPase-activating protein-binding proteins 1 and 2 (G3BPs) (two proteins targeted by tRF-27); RNA-immunoprecipitation (RIP) to confirm their bindings; co-immunoprecipitation (co-IP) and RNA-pulldown assay to determine the binding domains between G3BPs and tRF-27.tRF-27 bound to the nuclear transport factor 2 like domain(NTF2 domain) of G3BPs through a specific sequence. tRF-27 relied on G3BPs and NTF2 domain to increase trastuzumab tolerance. tRF-27 competed with lysosomal associated membrane protein 1(LAMP1) for NTF2 domain, thereby inhibiting lysosomal localization of G3BPs and tuberous sclerosis complex (TSC). Overexpression of tRF-27 inhibited phosphorylation of TSCs and promoted the activation of mechanistic target of rapamycin complex 1(MTORC1) to enhance cell proliferation and entice the resistance of HER2-positive breast cancer against trastuzumab.

Expression patterns and clinical value of key m6A RNA modification regulators in smoking patients with coronary artery disease.

Even though N6-methyladenosine (m6A) RNA modifications are increasingly being implicated in human disease, their mechanisms are not fully understood in smokers with coronary artery disease (CAD). Thirty m6A-related regulators' expression (MRRE) in CAD individuals (smokers and non-smokers) were analyzed from GEO. Support Vector Machine, random forest, and nomogram models were constructed to assess its clinical value. Consensus clustering, principal component analysis, and ssGSEA were used to construct a full picture of m6A-related regulators in smokers with CAD. Oxygen-glucose deprivation (OGD) and qRT-PCR were used to validate hypoxia's effect on MRRE. A comparison between smokers with CAD and controls revealed lower expression levels of RBM15B, YTHDC2, and ZC3H13. Based on three key MRREs, all models showed good clinical value, and smokers with CAD were divided into two distinct molecular subgroups. The correlations were found between key MRRE and the degree of immune infiltration. Three key MRREs in HUVECs and FMC84 mouse cardiomyocytes were reduced in the OGD group. Through hypoxia, smoking might reduce the expression levels of RBM15B, YTHDC2, and ZC3H13 in smokers with CAD. Our findings provide an important theoretical basis for the treatment of smokers with CAD.

SARS-CoV-2 nucleocapsid protein promotes self-deacetylation by inducing HDAC6 to facilitate viral replication.

BACKGROUND: The global outbreak of COVID-19 caused by the SARS-CoV-2 has led to millions of deaths. This unanticipated emergency has prompted virologists across the globe to delve deeper into the intricate dynamicity of the host-virus interface with an aim to identify antiviral targets and elucidate host and viral determinants of severe disease. AIM: The present study was undertaken to analyse the role of histone deacetylase 6 (HDAC6) in regulating SARS-CoV-2 infection. RESULTS: Gradual increase in HDAC6 expression was observed in different SARS-CoV-2-permissive cell lines following SARS-CoV-2 infection. The SARS-CoV-2 nucleocapsid protein (N protein) was identified as the primary viral factor responsible for upregulating HDAC6 expression. Downregulation of HDAC6 using shRNA or a specific inhibitor tubacin resulted in reduced viral replication suggesting proviral role of its deacetylase activity. Further investigations uncovered the interaction of HDAC6 with stress granule protein G3BP1 and N protein during infection. HDAC6-mediated deacetylation of SARS-CoV-2 N protein was found to be crucial for its association with G3BP1. CONCLUSION: This study provides valuable insights into the molecular mechanisms underlying the disruption of cytoplasmic stress granules during SARS-CoV-2 infection and highlights the significance of HDAC6 in the process.

High throughput screening for SARS-CoV-2 helicase inhibitors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for nearly 7 million deaths worldwide since its outbreak in late 2019. Even with the rapid development and production of vaccines and intensive research, there is still a huge need for specific anti-viral drugs that address the rapidly arising new variants. To address this concern, the National Institute of Allergy and Infectious Diseases (NIAID) established nine Antiviral Drug Discovery (AViDD) Centers, tasked with exploring approaches to target pathogens with pandemic potential, including SARS-CoV-2. In this study, we sought inhibitors of SARS-CoV2 non-structural protein 13 (nsP13) as potential antivirals, first developing a HTS-compatible assay to measure SARS-CoV2 nsP13 helicase activity. Here we present our effort in implementing the assay in a 1,536 well-plate format and in identifying nsP13 inhibitor hit compounds from a ∼650,000 compound library. The primary screen was robust (average Z' = 0.86 ± 0.05) and resulted in 7,009 primary hits. 1,763 of these compounds upon repeated retests were further confirmed, showing consistent inhibition. Following in-silico analysis, an additional orthogonal assay and titration assays, we identified 674 compounds with IC50 <10 µM. We confirmed activity of independent compound batches from de novo powders while also incorporating multiple counterscreen assays. Our study highlights the potential of this assay for use on HTS platforms to discover novel compounds inhibiting SARS-CoV2 nsP13, which merit further development as an effective SARS-CoV2 antiviral.

RH20, a phase-separated RNA helicase protein, facilitates plant resistance to viruses.

RNA silencing, a conserved gene regulatory mechanism, is critical for host resistance to viruses. Liquid-liquid phase separation (LLPS) is an important mechanism in regulating various biological processes. Emerging studies suggest RNA helicases play important roles in microRNA (miRNA) production through LLPS. In this study, we investigated the functional role of RNA helicase 20 (RH20), a DDX5 homolog in Arabidopsis thaliana, in RNA silencing and plant resistance to viruses. Our findings reveal that RH20 localizes in both the cytoplasm and nucleus, with puncta formation in the cytoplasm exhibiting liquid-liquid phase separation behavior. We demonstrate that RH20 plays positive roles in plant immunity against viruses. Further study showed that RH20 interacts with Argonaute 2 (AGO2), a key component of the RNA silencing pathway. Moreover, RH20 promotes the accumulation of both endogenous and exogenous small RNAs (sRNAs). Overall, our study identifies RH20 as a novel phase separation protein that interacting with AGO2, influencing sRNAs accumulation, and enhancing plant resistance to viruses.

DCAF7 Acts as A Scaffold to Recruit USP10 for G3BP1 Deubiquitylation and Facilitates Chemoresistance and Metastasis in Nasopharyngeal Carcinoma.

Despite docetaxel combined with cisplatin and 5-fluorouracil (TPF) being the established treatment for advanced nasopharyngeal carcinoma (NPC), there are patients who do not respond positively to this form of therapy. However, the mechanisms underlying this lack of benefit remain unclear. DCAF7 is identified as a chemoresistance gene attenuating the response to TPF therapy in NPC patients. DCAF7 promotes the cisplatin resistance and metastasis of NPC cells in vitro and in vivo. Mechanistically, DCAF7 serves as a scaffold protein that facilitates the interaction between USP10 and G3BP1, leading to the elimination of K48-linked ubiquitin moieties from Lys76 of G3BP1. This process helps prevent the degradation of G3BP1 via the ubiquitin‒proteasome pathway and promotes the formation of stress granule (SG)-like structures. Moreover, knockdown of G3BP1 successfully reversed the formation of SG-like structures and the oncogenic effects of DCAF7. Significantly, NPC patients with increased levels of DCAF7 showed a high risk of metastasis, and elevated DCAF7 levels are linked to an unfavorable prognosis. The study reveals DCAF7 as a crucial gene for cisplatin resistance and offers further understanding of how chemoresistance develops in NPC. The DCAF7-USP10-G3BP1 axis contains potential targets and biomarkers for NPC treatment.

A Repurposed Drug Interferes with Nucleic Acid to Inhibit the Dual Activities of Coronavirus Nsp13.

The recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlighted a critical need to discover more effective antivirals. While therapeutics for SARS-CoV-2 exist, its nonstructural protein 13 (Nsp13) remains a clinically untapped target. Nsp13 is a helicase responsible for unwinding double-stranded RNA during viral replication and is essential for propagation. Like other helicases, Nsp13 has two active sites: a nucleotide binding site that hydrolyzes nucleoside triphosphates (NTPs) and a nucleic acid binding channel that unwinds double-stranded RNA or DNA. Targeting viral helicases with small molecules, as well as the identification of ligand binding pockets, have been ongoing challenges, partly due to the flexible nature of these proteins. Here, we use a virtual screen to identify ligands of Nsp13 from a collection of clinically used drugs. We find that a known ion channel inhibitor, IOWH-032, inhibits the dual ATPase and helicase activities of SARS-CoV-2 Nsp13 at low micromolar concentrations. Kinetic and binding assays, along with computational and mutational analyses, indicate that IOWH-032 interacts with the RNA binding interface, leading to displacement of nucleic acid substrate, but not bound ATP. Evaluation of IOWH-032 with microbial helicases from other superfamilies reveals that it is selective for coronavirus Nsp13. Furthermore, it remains active against mutants representative of observed SARS-CoV-2 variants. Overall, this work provides a new inhibitor for Nsp13 and provides a rationale for a recent observation that IOWH-032 lowers SARS-CoV-2 viral loads in human cells, setting the stage for the discovery of other potent viral helicase modulators.

A novel signature constructed by cuproptosis-related RNA methylation regulators suggesting downregulation of YTHDC2 may induce cuproptosis resistance in colorectal cancer.

BACKGROUND: A newly identified type of cell death due to intracellular copper accumulation is known as cuproptosis and RNA methylation is a post-transcriptional modification mechanism, both of which perform vital roles in the immune microenvironment of colorectal cancer (CRC), but the link between the two needs more research. METHODS: TCGA database provided RNA-seq data and details clinically of CRC samples. Cuproptosis-related RNA methylation regulators (CRRMRs) were identified by correlation analysis. We screened 6 CRRMRs for prognostic model construction by employing LASSO-Cox regression analysis and calculated risk scores by CRRMRs (CuMS). GSE39582 and GSE38832 cohort were used as external validation sets. This research concentrated on the connection between the prognostic model and somatic mutation, anti-cancer drug sensitivity, immune infiltration, immune checkpoint expression. In addition, we investigated the differential expression of YTHDC2 in epithelial cell subpopulations by single-cell analysis with GSE166555, calculated cuproptosis scores and performed pathway enrichment. In vitro experiments were performed to explore the consequences of knockdown of YTHDC2 on CRC cell proliferation and migration, as well as changes in CRC cell viability in response to elesclomol after knockdown of YTHDC2. In vivo experiments, we constructed the cell line-derived xenograft model to further validate the results of the in vitro experiments. RESULTS: The prognosis of CRC can be predicted by CuMS, which GSE39582 and GSE38832 confirmed. Two CuMS groups showed different tumor mutation burden (TMB) and immune infiltration. CuMS was connected to emerging immune checkpoints CD47 and PVR, therefore, it can be clinically complementary to TMB and microsatellite instability (MSI) status. In single-cell analysis, a subpopulation of epithelial cells with high YTHDC2 expression had a high cuproptosis score. In vitro experiments, knocking down YTHDC2 promoted cell proliferation and migration in CRC, and weaken the inhibitory effect of elesclomol and elesclomol-Cu on cell viability, which in vivo experiments validated. CONCLUSION: We developed a prognostic model constructed by 6 CRRMRs to assess overall survival and immune microenvironment of CRC patients. YTHDC2 might regulate cuproptosis in multiple ways.

Variable Inhibition of DNA Unwinding Rates Catalyzed by the SARS-CoV-2 Helicase Nsp13 by Structurally Distinct Single DNA Lesions.

The SARS-CoV-2 helicase, non-structural protein 13 (Nsp13), plays an essential role in viral replication, translocating in the 5' → 3' direction as it unwinds double-stranded RNA/DNA. We investigated the impact of structurally distinct DNA lesions on DNA unwinding catalyzed by Nsp13. The selected lesions include two benzo[a]pyrene (B[a]P)-derived dG adducts, the UV-induced cyclobutane pyrimidine dimer (CPD), and the pyrimidine (6-4) pyrimidone (6-4PP) photolesion. The experimentally observed unwinding rate constants (kobs) and processivities (P) were examined. Relative to undamaged DNA, the kobs values were diminished by factors of up to ~15 for B[a]P adducts but only by factors of ~2-5 for photolesions. A minor-groove-oriented B[a]P adduct showed the smallest impact on P, which decreased by ~11% compared to unmodified DNA, while an intercalated one reduced P by ~67%. However, the photolesions showed a greater impact on the processivities; notably, the CPD, with the highest kobs value, exhibited the lowest P, which was reduced by ~90%. Our findings thus show that DNA unwinding efficiencies are lesion-dependent and most strongly inhibited by the CPD, leading to the conclusion that processivity is a better measure of DNA lesions' inhibitory effects than unwinding rate constants.

The RNA helicase DHX35 functions as a co-sensor for RIG-I-mediated innate immunity.

RNA helicases are involved in the innate immune response against pathogens, including bacteria and viruses; however, their mechanism in the human airway epithelial cells is still not fully understood. Here, we demonstrated that DEAH (Asp-Glu-Ala-His) box polypeptide 35 (DHX35), a member of the DExD/H (Asp-Glu-x-Asp/His)-box helicase family, boosts antiviral innate immunity in human airway epithelial cells. DHX35 knockdown attenuated the production of interferon-ß (IFN-ß), IL6, and CXCL10, whereas DHX35 overexpression increased their production. Upon stimulation, DHX35 was constitutively expressed, but it translocated from the nucleus into the cytosol, where it recognized cytosolic poly(I:C) and poly(dA:dT) via its HELICc domain. Mitochondrial antiviral signaling protein (MAVS) acted as an adaptor for DHX35 and interacted with the HELICc domain of DHX35 using amino acids 360-510. Interestingly, DHX35 interacted with retinoic acid-inducible gene 1 (RIG-I), enhanced the binding affinity of RIG-I with poly(I:C) and poly(dA:dT), and formed a signalsome with MAVS to activate interferon regulatory factor 3 (IRF3), NF-κB-p65, and MAPK signaling pathways. These results indicate that DHX35 not only acted as a cytosolic nucleic acid sensor but also synergized with RIG-I to enhance antiviral immunity in human airway epithelial cells. Our results demonstrate a novel molecular mechanism for DHX35 in RIG-I-mediated innate immunity and provide a novel candidate for drug and vaccine design to control viral infections in the human airway.

UPF1 plays critical roles in early B cell development.

The ATP-dependent RNA helicase UPF1 plays a crucial role in various mRNA degradation pathways, most importantly in nonsense-mediated mRNA decay (NMD). Here, we show that UPF1 is upregulated during the early stages of B cell development and is important for early B cell development in the bone marrow. B-cell-specific Upf1 deletion in mice severely impedes the early to late LPre-B cell transition, in which VH-DHJH recombination occurs at the Igh gene. Furthermore, UPF1 is indispensable for VH-DHJH recombination, without affecting DH-JH recombination. Intriguingly, the genetic pre-arrangement of the Igh gene rescues the differentiation defect in early LPre-B cells under Upf1 deficient conditions. However, differentiation is blocked again following Ig light chain recombination, leading to a failure in development into immature B cells. Notably, UPF1 interacts with and regulates the expression of genes involved in immune responses, cell cycle control, NMD, and the unfolded protein response in B cells. Collectively, our findings underscore the critical roles of UPF1 during the early LPre-B cell stage and beyond, thus orchestrating B cell development.