PARP1, DIDO3, and DHX9 Proteins Mutually Interact in Mouse Fibroblasts, with Effects on DNA Replication Dynamics, Senescence, and Oncogenic Transformation.

The regulated formation and resolution of R-loops is a natural process in physiological gene expression. Defects in R-loop metabolism can lead to DNA replication stress, which is associated with a variety of diseases and, ultimately, with cancer. The proteins PARP1, DIDO3, and DHX9 are important players in R-loop regulation. We previously described the interaction between DIDO3 and DHX9. Here, we show that, in mouse embryonic fibroblasts, the three proteins are physically linked and dependent on PARP1 activity. The C-terminal truncation of DIDO3 leads to the impairment of this interaction; concomitantly, the cells show increased replication stress and senescence. DIDO3 truncation also renders the cells partially resistant to in vitro oncogenic transformation, an effect that can be reversed by immortalization. We propose that PARP1, DIDO3, and DHX9 proteins form a ternary complex that regulates R-loop metabolism, preventing DNA replication stress and subsequent senescence.

DEAD-Box RNA Helicase DDX47 Maintains Midgut Homeostasis in Locusta migratoria.

Tissue homeostasis is critical for maintaining organ shape, size, and function. The condition is regulated by the balance between the generation of new cells and the loss of senescent cells, and it involves many factors and mechanisms. The midgut, an important part of the intestinal tract, is responsible for digestion and nutrient absorption in insects. LmDDX47, the ortholog of DEAD-box helicase 47 from Locusta migratoria, is indispensable for sustaining a normal midgut in the nymphs. However, the underlying cellular and molecular mechanisms remain to be elucidated. In this study, LmDDX47 knockdown resulted in atrophy of the midgut and gastric cecum in both nymph and adult locusts. After LmDDX47 knockdown, the number of regenerative and columnar cells in the midgut was significantly reduced, and cell death was induced in columnar tissue. LmDDX47 was localized to the nucleolus; this was consistent with the reduction in 18S rRNA synthesis in the LmDDX47 knockdown group. In addition, the acetylation and crotonylation levels of midgut proteins were significantly increased. Therefore, LmDDX47 could be a key regulator of midgut homeostasis, regulating 18S rRNA synthesis as well as protein acetylation and crotonylation in the migratory locust.

G-Quadruplexes and the DNA/RNA helicase DHX36 in health, disease, and aging.

G-Quadruplex (G4) DNA (G4 DNA) and RNA (G4 RNA) are secondary nucleic acid structures that have multiple roles in vital cellular processes. G4 DNA- and RNA-binding proteins and unwinding helicases associate with and regulate G4s during virtually all processes that involve DNA and RNA. DEAH-Box helicase 36 (DHX36), a member of the large DExD/H box helicase family, enzymatically unwinds both G4 DNA and G4 RNA. By exerting its G4 helicase function, DHX36 regulates transcription, genomic stability, telomere maintenance, translation and RNA metabolism. This review will provide an overview of G4s and DHX36, including DHX36's potential role in neuronal development and neurodegeneration. We conclude with a discussion of the possible functions of G4s and DHX36 in the aging brain.

DDX46 accelerates the proliferation of glioblastoma by activating the MAPK-p38 signaling.

PURPOSE: To analyze the influence of DDX46 on the proliferative and migratory potentials of glioblastoma (GBM). METHODS: Differential levels of DDX46 in GBM cases and controls were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. By intervening DDX46 in U87 and U251 cells, proliferative and migratory changes were determined by colony formation assay, 5-Ethynyl-2'- deoxyuridine (EdU) assay and Transwell assay, respectively. Protein levels of p-p38, p38, cyclin D1 and MMP7 in GBM cells intervened by DDX46 or the inhibitor of p38 MAPK were detected. RESULTS: DDX46 was upregulated in GBM cases. Knockdown of DDX46 attenuated the proliferative capacity of GBM cells, and its overexpression enhanced the proliferative rate. The migratory capacity of GBM was not affected by DDX46. Overexpression of DDX46 upregulated p-p38 and cyclin D1 in GBM cells. The regulatory effect of DDX46 on GBM proliferation could be partially reversed by the treatment of doramapimod. CONCLUSIONS: DDX46 is upregulated in GBM, which strengthens the proliferative capacity of GBM by activating the MAPK-p38 signaling.

Impact of DICER1 and DROSHA on the Angiogenic Capacity of Human Endothelial Cells.

RNAi-mediated knockdown of DICER1 and DROSHA, enzymes critically involved in miRNA biogenesis, has been postulated to affect the homeostasis and the angiogenic capacity of human endothelial cells. To re-evaluate this issue, we reduced the expression of DICER1 or DROSHA by RNAi-mediated knockdown and subsequently investigated the effect of these interventions on the angiogenic capacity of human umbilical vein endothelial cells (HUVEC) in vitro (proliferation, migration, tube formation, endothelial cell spheroid sprouting) and in a HUVEC xenograft assay in immune incompetent NSGTM mice in vivo. In contrast to previous reports, neither knockdown of DICER1 nor knockdown of DROSHA profoundly affected migration or tube formation of HUVEC or the angiogenic capacity of HUVEC in vivo. Furthermore, knockdown of DICER1 and the combined knockdown of DICER1 and DROSHA tended to increase VEGF-induced BrdU incorporation and induced angiogenic sprouting from HUVEC spheroids. Consistent with these observations, global proteomic analyses showed that knockdown of DICER1 or DROSHA only moderately altered HUVEC protein expression profiles but additively reduced, for example, expression of the angiogenesis inhibitor thrombospondin-1. In conclusion, global reduction of miRNA biogenesis by knockdown of DICER1 or DROSHA does not inhibit the angiogenic capacity of HUVEC. Further studies are therefore needed to elucidate the influence of these enzymes in the context of human endothelial cell-related angiogenesis.

The DEAD-box RNA helicase RhlE2 is a global regulator of Pseudomonas aeruginosa lifestyle and pathogenesis.

RNA helicases perform essential housekeeping and regulatory functions in all domains of life by binding and unwinding RNA molecules. The bacterial RhlE-like DEAD-box RNA helicases are among the least well studied of these enzymes. They are widespread especially among Proteobacteria, whose genomes often encode multiple homologs. The significance of the expansion and diversification of RhlE-like proteins for bacterial fitness has not yet been established. Here, we study the two RhlE homologs present in the opportunistic pathogen Pseudomonas aeruginosa. We show that, in the course of evolution, RhlE1 and RhlE2 have diverged in their biological functions, molecular partners and RNA-dependent enzymatic activities. Whereas RhlE1 is mainly needed for growth in the cold, RhlE2 also acts as global post-transcriptional regulator, affecting the level of hundreds of cellular transcripts indispensable for both environmental adaptation and virulence. The global impact of RhlE2 is mediated by its unique C-terminal extension, which supports the RNA unwinding activity of the N-terminal domain as well as an RNA-dependent interaction with the RNase E endonuclease and the cellular RNA degradation machinery. Overall, our work reveals how the functional and molecular divergence between two homologous RNA helicases can contribute to bacterial fitness and pathogenesis.

RNA Helicase A/DHX9 Forms Unique Cytoplasmic Antiviral Granules That Restrict Oncolytic Myxoma Virus Replication in Human Cancer Cells.

RNA helicase A/DHX9 is required for diverse RNA-related essential cellular functions and antiviral responses and is hijacked by RNA viruses to support their replication. Here, we show that during the late replication stage in human cancer cells of myxoma virus (MYXV), a member of the double-stranded DNA (dsDNA) poxvirus family that is being developed as an oncolytic virus, DHX9, forms unique granular cytoplasmic structures, which we named "DHX9 antiviral granules." These DHX9 antiviral granules are not formed if MYXV DNA replication and/or late protein synthesis is blocked. When formed, DHX9 antiviral granules significantly reduced nascent protein synthesis in the MYXV-infected cancer cells. MYXV late gene transcription and translation were also significantly compromised, particularly in nonpermissive or semipermissive human cancer cells where MYXV replication is partly or completely restricted. Directed knockdown of DHX9 significantly enhanced viral late protein synthesis and progeny virus formation in normally restrictive cancer cells. We further demonstrate that DHX9 is not a component of the canonical cellular stress granules. DHX9 antiviral granules are induced by MYXV, and other poxviruses, in human cells and are associated with other known cellular components of stress granules, dsRNA and virus encoded dsRNA-binding protein M029, a known interactor with DHX9. Thus, DHX9 antiviral granules function by hijacking poxviral elements needed for the cytoplasmic viral replication factories. These results demonstrate a novel antiviral function for DHX9 that is recruited from the nucleus into the cytoplasm, and this step can be exploited to enhance oncolytic virotherapy against the subset of human cancer cells that normally restrict MYXV. IMPORTANCE The cellular DHX9 has both proviral and antiviral roles against diverse RNA and DNA viruses. In this article, we demonstrate that DHX9 can form unique antiviral granules in the cytoplasm during myxoma virus (MYXV) replication in human cancer cells. These antiviral granules sequester viral proteins and reduce viral late protein synthesis and thus regulate MYXV, and other poxviruses, that replicate in the cytoplasm. In addition, we show that in the absence of DHX9, the formation of DHX9 antiviral granules can be inhibited, which significantly enhanced oncolytic MYXV replication in human cancer cell lines where the virus is normally restricted. Our results also show that DHX9 antiviral granules are formed after viral infection but not by common nonviral cellular stress inducers. Thus, our study suggests that DHX9 has antiviral activity in human cancer cells, and this pathway can be targeted for enhanced activity of oncolytic poxviruses against even restrictive cancer cells.

The RNA helicase DDX5 promotes viral infection via regulating N6-methyladenosine levels on the DHX58 and NFκB transcripts to dampen antiviral innate immunity.

Multi-functional DEAD-box helicase 5 (DDX5), which is important in transcriptional regulation, is hijacked by diverse viruses to facilitate viral replication. However, its regulatory effect in antiviral innate immunity remains unclear. We found that DDX5 interacts with the N6-methyladenosine (m6A) writer METTL3 to regulate methylation of mRNA through affecting the m6A writer METTL3-METTL14 heterodimer complex. Meanwhile, DDX5 promoted the m6A modification and nuclear export of transcripts DHX58, p65, and IKKγ by binding conserved UGCUGCAG element in innate response after viral infection. Stable IKKγ and p65 transcripts underwent YTHDF2-dependent mRNA decay, whereas DHX58 translation was promoted, resulting in inhibited antiviral innate response by DDX5 via blocking the p65 pathway and activating the DHX58-TBK1 pathway after infection with RNA virus. Furthermore, we found that DDX5 suppresses antiviral innate immunity in vivo. Our findings reveal that DDX5 serves as a negative regulator of innate immunity by promoting RNA methylation of antiviral transcripts and consequently facilitating viral propagation.

DDX3 depletion represses translation of mRNAs with complex 5' UTRs.

DDX3 is an RNA chaperone of the DEAD-box family that regulates translation. Ded1, the yeast ortholog of DDX3, is a global regulator of translation, whereas DDX3 is thought to preferentially affect a subset of mRNAs. However, the set of mRNAs that are regulated by DDX3 are unknown, along with the relationship between DDX3 binding and activity. Here, we use ribosome profiling, RNA-seq, and PAR-CLIP to define the set of mRNAs that are regulated by DDX3 in human cells. We find that while DDX3 binds highly expressed mRNAs, depletion of DDX3 particularly affects the translation of a small subset of the transcriptome. We further find that DDX3 binds a site on helix 16 of the human ribosomal rRNA, placing it immediately adjacent to the mRNA entry channel. Translation changes caused by depleting DDX3 levels or expressing an inactive point mutation are different, consistent with different association of these genetic variant types with disease. Taken together, this work defines the subset of the transcriptome that is responsive to DDX3 inhibition, with relevance for basic biology and disease states where DDX3 is altered.

Extending the L1 region in canonical double-stranded RNA-binding domains impairs their functions.

A large number of proteins involved in RNA metabolism possess a double-stranded RNA-binding domain (dsRBD), whose sequence variations and functional versatilities are still being recognized. All dsRBDs have a similar structural fold: α1-L1-ß1-L2-ß2-L3-ß3-L4-α2 (α represents an α-helix, ß a ß-sheet, and L a loop conformation between the well-defined secondary structures). Our recent work revealed that the dsRBD in Drosha, which is involved in animal microRNA (miRNA) biogenesis, differs from other dsRBDs by containing a short insertion in its L1 region and that this insertion is important for Drosha function. We asked why the same insertion is excluded in all other dsRBDs and proposed that a longer L1 may be detrimental to their functions. In this study, to test this hypothesis, we inserted the Drosha sequence into several well-known dsRBDs from various organisms. Gel mobility shift assay demonstrated that L1 extension invariably reduced RNA binding by these dsRBDs. In addition, such a mutation in Dicer, another protein involved in miRNA biogenesis, impaired Dicer's ability to process miRNAs, which led to de-repression of reporter expression, in human cells. Taken together, our results add to the growing appreciation of the diversity in dsRBDs and suggest that dsRBDs have intricate structures and functions that are sensitive to perturbations in the L1 region.

Happy Birthday: 30 Years of RNA Helicases.

RNA helicases are ubiquitous, highly conserved RNA-binding enzymes that use the energy derived from the hydrolysis of nucleoside triphosphate to modify the structure of RNA molecules and/or the functionality of ribonucleoprotein complexes. Ultimately, the action of RNA helicases results in changes in gene expression that allow the cell to perform crucial functions. In this chapter, we review established and emerging concepts for DEAD-box and DExH-box RNA helicases. We mention examples from both eukaryotic and prokaryotic systems, in order to highlight common themes and specific actions.

[High-level androgen enhances the differentiation of hESCs into male primordial germ cells].

OBJECTIVE: To observe the effects of different concentrations of testosterone on the differentiation of human embryonic stem cells (hESCs) into early male germ cells and investigate the potential impact of high-level androgen exposure in early pregnancy in women with polycystic ovary syndrome (PCOS) on the fertility and primordial germ cell reserve of the male offspring in adulthood. METHODS: We used 2 µmol/L retinoic acid to induce the differentiation of hESCs (46, XY) into male germ cells in vitro and meanwhile treated them with testosterone (T) at 0 mol/L, 3×10－7 mol/L, 5×10－7 mol/L, 15×10－7 mol/L, 45×10－7 mol/L, and 135×10－7 mol/L, respectively. We collected the cell samples at 0, 4, 7 and 14 days to determine the expressions of the specific genes and compare the differentiation process and efficiency of the male germ cells in different stages. RESULTS: There was no difference in the morphology of the hESCs treated with different concentrations of testosterone in the same differentiation stage. The expression of the marker gene DAZL in the primordial germ cells peaked on the 4th day of differentiation, significantly higher in the 15×10－7, 45×10－7 and 135×10－7 mol/L groups than in the 3×10－7 mol/L group (P < 0.05), and that of the specific gene SCP3 in the early-meiosis germ cells began to increase on the same day, more significantly in the 45×10－7mol/L than in the 3×10－7 mol/L and 5×10－7 mol/L groups (P < 0.01), and peaked on the 7th day, dramatically higher in the 15×10－7, 45×10－7 and 135×10－7 mol/L groups than in the 3×10－7 mol/L group (P < 0.01). Immunofluorescence staining and flow cytometry showed a T concentration-dependent increase in the expression of DAZL at 4 days and those of SCP3 and VASA at 7 days. Moreover, the expression of the androgen receptor (AR) in the hESCs began to rise on the 4th day and kept going up till the 14th day, higher in the high-concentration than in the low-concentration T groups in the same stage of differentiation, though with no statistically significant difference (P > 0.05). CONCLUSIONS: Exposure to high-level androgen during the differentiation of hESCs into early male germ cells can induce earlier expression of AR and earlier differentiation of hESCs into early male germ cells, which may result in insufficient reserve of male primary germ cells in the male offspring of PCOS women and affect their fertility after adulthood. hESCs can be used as an in vitro model to study the effects of intrauterine hyperandrogen on the reproductive development of male offspring in PCOS patients, which is also contributive to researches on the etiology of male infertility.

Role reversal of functional identity in host factors: Dissecting features affecting pro-viral versus antiviral functions of cellular DEAD-box helicases in tombusvirus replication.

Positive-stranded (+)RNA viruses greatly exploit host cells to support viral replication. However, unlike many other pathogens, (+)RNA viruses code for only a limited number of genes, making them highly dependent on numerous co-opted host factors for supporting viral replication and other viral processes during their infections. This excessive dependence on subverted host factors, however, renders (+)RNA viruses vulnerable to host restriction factors that could block virus replication. Interestingly, cellular ATP-dependent DEAD-box RNA helicases could promote or inhibit the replication of Tomato bushy stunt virus (TBSV) replication. However, it is currently unknown what features make a particular DEAD-box helicase either pro-viral or antiviral. In this work, we succeeded in reversing the viral function of the antiviral DDX17-like RH30 DEAD-box helicase by converting it to a pro-viral helicase. We also turned the pro-viral DDX3-like RH20 helicase into an antiviral helicase through deletion of a unique N-terminal domain. We demonstrate that in the absence of the N-terminal domain, the core helicase domain becomes unhinged, showing altered specificity in unwinding viral RNA duplexes containing cis-acting replication elements. The discovery of the sequence plasticity of DEAD-box helicases that can alter recognition of different cis-acting RNA elements in the viral genome illustrates the evolutionary potential of RNA helicases in the arms race between viruses and their hosts, including key roles of RNA helicases in plant innate immunity. Overall, these findings open up the possibility to turn the pro-viral host factors into antiviral factors, thus increasing the potential antiviral arsenal of the host for the benefit of agriculture and health science.

LncRNA DDX11-AS1: a novel oncogene in human cancer.

Long noncoding RNA (lncRNA) is a newly identified type of noncoding RNA with a length of more than 200 nucleotides. The latest research shows that lncRNAs play important roles in the occurrence and development of human tumours by acting both as carcinogenic genes and as tumour suppressor genes. LncRNAs plays a role in various biological processes, such as cell growth, apoptosis, migration and invasion. The newly discovered lncRNA DDX11-AS1 is abnormally highly expressed in various malignant tumours, such as hepatocellular carcinoma, colorectal cancer, osteosarcoma, bladder cancer, NSCLC and gastric cancer. DDX11-AS1 mainly regulates the expression of related genes through direct or indirect ways to perform its functions in carcinogenicity. These results indicate that DDX11-AS1 may be a marker or therapeutic target of tumours. This review summarizes the biological function and mechanism of DDX11-AS1 in the process of tumour development.

DDX5 promotes oncogene C3 and FABP1 expressions and drives intestinal inflammation and tumorigenesis.

Tumorigenesis in different segments of the intestinal tract involves tissue-specific oncogenic drivers. In the colon, complement component 3 (C3) activation is a major contributor to inflammation and malignancies. By contrast, tumorigenesis in the small intestine involves fatty acid-binding protein 1 (FABP1). However, little is known of the upstream mechanisms driving their expressions in different segments of the intestinal tract. Here, we report that the RNA-binding protein DDX5 binds to the mRNA transcripts of C3 and Fabp1 to augment their expressions posttranscriptionally. Knocking out DDX5 in epithelial cells protected mice from intestinal tumorigenesis and dextran sodium sulfate (DSS)-induced colitis. Identification of DDX5 as a common upstream regulator of tissue-specific oncogenic molecules provides an excellent therapeutic target for intestinal diseases.

DICER regulates the expression of major satellite repeat transcripts and meiotic chromosome segregation during spermatogenesis.

Constitutive heterochromatin at the pericentric regions of chromosomes undergoes dynamic changes in its epigenetic and spatial organization during spermatogenesis. Accurate control of pericentric heterochromatin is required for meiotic cell divisions and production of fertile and epigenetically intact spermatozoa. In this study, we demonstrate that pericentric heterochromatin is expressed during mouse spermatogenesis to produce major satellite repeat (MSR) transcripts. We show that the endonuclease DICER localizes to the pericentric heterochromatin in the testis. Furthermore, DICER forms complexes with MSR transcripts, and their processing into small RNAs is compromised in Dicer1 knockout mice leading to an elevated level of MSR transcripts in meiotic cells. We also show that defective MSR forward transcript processing in Dicer1 cKO germ cells is accompanied with reduced recruitment of SUV39H2 and H3K9me3 to the pericentric heterochromatin and meiotic chromosome missegregation. Altogether, our results indicate that the physiological role of DICER in maintenance of male fertility extends to the regulation of pericentric heterochromatin through direct targeting of MSR transcripts.

Arabidopsis SMN2/HEN2, Encoding DEAD-Box RNA Helicase, Governs Proper Expression of the Resistance Gene SMN1/RPS6 and Is Involved in Dwarf, Autoimmune Phenotypes of mekk1 and mpk4 Mutants.

In Arabidopsis thaliana, a mitogen-activated protein kinase pathway, MEKK1-MKK1/MKK2-MPK4, is important for basal resistance and disruption of this pathway results in dwarf, autoimmune phenotypes. To elucidate the complex mechanisms activated by the disruption of this pathway, we have previously developed a mutant screening system based on a dwarf autoimmune line that overexpressed the N-terminal regulatory domain of MEKK1. Here, we report that the second group of mutants, smn2, had defects in the SMN2 gene, encoding a DEAD-box RNA helicase. SMN2 is identical to HEN2, whose function is vital for the nuclear RNA exosome because it provides non-ribosomal RNA specificity for RNA turnover, RNA quality control and RNA processing. Aberrant SMN1/RPS6 transcripts were detected in smn2 and hen2 mutants. Disease resistance against Pseudomonas syringae pv. tomato DC3000 (hopA1), which is conferred by SMN1/RPS6, was decreased in smn2 mutants, suggesting a functional connection between SMN1/RPS6 and SMN2/HEN2. We produced double mutants mekk1smn2 and mpk4smn2 to determine whether the smn2 mutations suppress the dwarf, autoimmune phenotypes of the mekk1 and mpk4 mutants, as the smn1 mutations do. As expected, the mekk1 and mpk4 phenotypes were suppressed by the smn2 mutations. These results suggested that SMN2 is involved in the proper function of SMN1/RPS6. The Gene Ontology enrichment analysis using RNA-seq data showed that defense genes were downregulated in smn2, suggesting a positive contribution of SMN2 to the genome-wide expression of defense genes. In conclusion, this study provides novel insight into plant immunity via SMN2/HEN2, an essential component of the nuclear RNA exosome.

The RNA helicase DDX5 supports mitochondrial function in small cell lung cancer.

DEAD-box helicase 5 (DDX5) is a founding member of the DEAD-box RNA helicase family, a group of enzymes that regulate ribonucleoprotein formation and function in every aspect of RNA metabolism, ranging from synthesis to decay. Our laboratory previously found that DDX5 is involved in energy homeostasis, a process that is altered in many cancers. Small cell lung cancer (SCLC) is an understudied cancer type for which effective treatments are currently unavailable. Using an array of methods, including short hairpin RNA-mediated gene silencing, RNA and ChIP sequencing analyses, and metabolite profiling, we show here that DDX5 is overexpressed in SCLC cell lines and that its down-regulation results in various metabolic and cellular alterations. Depletion of DDX5 resulted in reduced growth and mitochondrial dysfunction in the chemoresistant SCLC cell line H69AR. The latter was evidenced by down-regulation of genes involved in oxidative phosphorylation and by impaired oxygen consumption. Interestingly, DDX5 depletion specifically reduced intracellular succinate, a TCA cycle intermediate that serves as a direct electron donor to mitochondrial complex II. We propose that the oncogenic role of DDX5, at least in part, manifests as up-regulation of respiration supporting the energy demands of cancer cells.

Condensation of Ded1p Promotes a Translational Switch from Housekeeping to Stress Protein Production.

Cells sense elevated temperatures and mount an adaptive heat shock response that involves changes in gene expression, but the underlying mechanisms, particularly on the level of translation, remain unknown. Here we report that, in budding yeast, the essential translation initiation factor Ded1p undergoes heat-induced phase separation into gel-like condensates. Using ribosome profiling and an in vitro translation assay, we reveal that condensate formation inactivates Ded1p and represses translation of housekeeping mRNAs while promoting translation of stress mRNAs. Testing a variant of Ded1p with altered phase behavior as well as Ded1p homologs from diverse species, we demonstrate that Ded1p condensation is adaptive and fine-tuned to the maximum growth temperature of the respective organism. We conclude that Ded1p condensation is an integral part of an extended heat shock response that selectively represses translation of housekeeping mRNAs to promote survival under conditions of severe heat stress.

The res (restored cell structure by salinity) tomato mutant reveals the role of the DEAD-box RNA helicase SlDEAD39 in plant development and salt response.

Increasing evidences highlight the importance of DEAD-box RNA helicases in plant development and stress responses. In a previous study, we characterized the tomato res mutant (restored cell structure by salinity), showing chlorosis and development alterations that reverted under salt-stress conditions. Map-based cloning demonstrates that RES gene encodes SlDEAD39, a chloroplast-targeted DEAD-box RNA helicase. Constitutive expression of SlDEAD39 complements the res mutation, while the silencing lines had a similar phenotype than res mutant, which is also reverted under salinity. Functional analysis of res mutant proved SlDEAD39 is involved in the in vivo processing of the chloroplast, 23S rRNA, at the hidden break-B site, a feature also supported by in vitro binding experiments of the protein. In addition, our results show that other genes coding for chloroplast-targeted DEAD-box proteins are induced by salt-stress, which might explain the rescue of the res mutant phenotype. Interestingly, salinity restored the phenotype of res adult plants by increasing their sugar content and fruit yield. Together, these results propose an unprecedented role of a DEAD-box RNA helicase in regulating plant development and stress response through the proper ribosome and chloroplast functioning, which, in turn, represents a potential target to improve salt tolerance in tomato crops.