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Neurodevelopmental disorders (NDDs) represent a large group of disorders with an onset in the neonatal or early childhood period; NDDs include intellectual disability (ID), autism spectrum disorders (ASD), attention deficit hyperactivity disorders (ADHD), seizures, various motor disabilities and abnormal muscle tone. Among the many underlying Mendelian genetic causes for these conditions, genes coding for proteins involved in all aspects of the gene expression pathway, ranging from transcription, splicing, translation to the eventual RNA decay, feature rather prominently. Here we focus on two large families of RNA helicases (DEAD- and DExH-box helicases). Genetic variants in the coding genes for several helicases have recently been shown to be associated with NDD. We address genetic constraints for helicases, types of pathological variants which have been discovered and discuss the biological pathways in which the affected helicase proteins are involved. [ABSTRACT FROM AUTHOR]

Introduction: Drugs that target reactive oxygen species (ROS) metabolism have progressed the treatment of pancreatic cancer treatment, yet their efficacy remains poor because of the adaptation of cancer cells to high concentration of ROS. Cells cope with ROS by recognizing 8-oxoguanine residues and processing severely oxidized RNA, which make it feasible to improve the efficacy of ROS-modulating drugs in pancreatic cancer by targeting 8-oxoguanine regulators.Methods: Poly(rC)-binding protein 1 (PCBP1) was identified as a potential oncogene in pancreatic cancer through datasets of The Cancer Genome Atlas (TCGA) project and Gene Expression Omnibus (GEO). High-throughput virtual screening was used to screen out potential inhibitors for PCBP1. Computational molecular dynamics simulations was used to verify the stable interaction between the two compounds and PCBP1 and their structure–activity relationships. In vitro experiments were performed for functional validation of silychristin.Results: In this study, we identified PCBP1 as a potential oncogene in pancreatic cancer. By applying high-throughput virtual screening, we identified Compound 102 and Compound 934 (silychristin) as potential PCBP1 inhibitors. Computational molecular dynamics simulations and virtual alanine mutagenesis verified the structure–activity correlation between PCBP1 and the two identified compounds. These two compounds interfere with the PCBP1–RNA interaction and impair the ability of PCBP1 to process RNA, leading to intracellular R loop accumulation. Compound 934 synergized with ROS agent hydrogen peroxide to strongly improve induced cell death in pancreatic cancer cells.Discussion: Our results provide valuable insights into the development of drugs that target PCBP1 and identified promising synergistic agents for ROS-modulating drugs in pancreatic cancer.

Neurodevelopmental disorders (NDDs) represent a large group of disorders with an onset in the neonatal or early childhood period; NDDs include intellectual disability (ID), autism spectrum disorders (ASD), attention deficit hyperactivity disorders (ADHD), seizures, various motor disabilities and abnormal muscle tone. Among the many underlying Mendelian genetic causes for these conditions, genes coding for proteins involved in all aspects of the gene expression pathway, ranging from transcription, splicing, translation to the eventual RNA decay, feature rather prominently. Here we focus on two large families of RNA helicases (DEAD- and DExH-box helicases). Genetic variants in the coding genes for several helicases have recently been shown to be associated with NDD. We address genetic constraints for helicases, types of pathological variants which have been discovered and discuss the biological pathways in which the affected helicase proteins are involved.

An R-loop is a nucleic acid structure consisting of a DNA : RNA hybrid and single-stranded DNA. It is formed physiologically in normal cells and is involved in transcription, replication, and gene rearrangement; in particular, it has multiple roles including in mitochondrial DNA replication and class switch recombination of immunoglobulin genes in B cells. However, accumulating evidence indicates aberrant R-loop formation in various malignancies, including hematopoietic neoplasms. The accumulation of such inappropriate R-loops can cause conflicts between transcription and DNA replication. This exacerbates genomic instability through the generation of DNA replication stress, that, in turn, leads to cellular phenotypic changes and disease progression. When RNAs are synthesized during transcription they hybridize with template DNA in cis, giving rise to R-loops. In addition, it was recently revealed that noncoding RNAs also form R-loops when bound to genomic DNA in trans. Together with such observations, new roles for the R-loop in disease development have been proposed. The relationship between inflammation and the R-loop has also attracted much attention. In this review, we will focus on the mechanisms of R-loop formation in various hematopoietic neoplasms and introduce the important findings from recent studies. Therapeutic concepts for targeting R-loop accumulation in hematopoietic neoplasms will also be discussed.

R-loop, an RNA-DNA hybrid structure, arises as a transcriptional by-product and has been implicated in DNA damage and genomic instability when excessive R-loop is accumulated. Although previous study demonstrated that R-loop is associated with ten-eleven translocation (Tet) proteins, which oxidize 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), the sixth base of DNA. However, the relationship between R-loop and DNA 5hmC modification remains unclear. In this study, we found that chronic restraint stress increased R-loop accumulation and decreased 5hmC modification in the prefrontal cortex (PFC) of the stressed mice. The increase of DNA 5hmC modification by vitamin C was accompanied with the decrease of R-loop levels; on the contrary, the decrease of DNA 5hmC modification by a small compound SC-1 increased the R-loop levels, indicating that 5hmC modification inversely regulates R-loop accumulation. Further, we showed that Tet deficiency-induced reduction of DNA 5hmC promoted R-loop accumulation. In addition, Tet proteins immunoprecipitated with Non- POU domain-containing octamer-binding (NONO) proteins. The deficiency of Tet proteins or NONO increased R-loop levels, but silencing Tet proteins and NONO did not further increase the increase accumulation, suggesting that NONO and Tet proteins formed a complex to inhibit R-loop formation. It was worth noting that NONO protein levels decreased in the PFC of stressed mice with R-loop accumulation. The administration of antidepressant fluoxetine to stressed mice increased NONO protein levels, and effectively decreased R-loop accumulation and DNA damage. In conclusion, we showed that DNA 5hmC modification negatively regulates R-loop accumulation by the NONO-Tet complex under stress. Our findings provide potential therapeutic targets for depression. [ABSTRACT FROM AUTHOR]

R-loops are three-stranded nucleic acid structures consisting of an RNA:DNA hybrid and a displaced DNA strand. While R-loops pose a potential threat to genome integrity, they constitute 5% of the human genome. The role of R-loops in transcriptional regulation, DNA replication, and chromatin signature is becoming increasingly clear. R-loops are associated with various histone modifications, suggesting that they may modulate chromatin accessibility. To potentially harness transcription-coupled repair mechanisms in the germline, nearly the entire genome is expressed during the early stages of male gametogenesis in mammals, providing ample opportunity for the formation of a transcriptomedependent R-loop landscape in male germ cells. In this study, our data demonstrated the presence of R-loops in fully mature human and bonobo sperm heads and their partial correspondence to transcribed regions and chromatin structure, which is massively reorganized from mainly histone to mainly protamine-packed chromatin in mature sperm. The sperm R-loop landscape resembles characteristic patterns of somatic cells. Surprisingly, we detected R-loops in both residual histone and protamine-packed chromatin and localize them to still-active retroposons, ALUs and SINE-VNTR-ALUs (SVAs), the latter has recently arisen in hominoid primates. We detected both evolutionarily conserved and species-specific localizations. Comparing our DNARNA immunoprecipitation (DRIP) data with published DNA methylation and histone chromatin immunoprecipitation (ChIP) data, we hypothesize that R-loops epigenetically reduce methylation of SVAs. Strikingly, we observe a strong influence of R-loops on the transcriptomes of zygotes from early developmental stages before zygotic genome activation. Overall, these findings suggest that chromatin accessibility influenced by R-loops may represent a system of inherited gene regulation. [ABSTRACT FROM AUTHOR]

R-loops are three-stranded nucleic acid structures consisting of an RNA:DNA hybrid and a displaced DNA strand. While R-loops pose a potential threat to genome integrity, they constitute 5% of the human genome. The role of R-loops in transcriptional regulation, DNA replication, and chromatin signature is becoming increasingly clear. R-loops are associated with various histone modifications, suggesting that they may modulate chromatin accessibility. To potentially harness transcription-coupled repair mechanisms in the germline, nearly the entire genome is expressed during the early stages of male gametogenesis in mammals, providing ample opportunity for the formation of a transcriptome-dependent R-loop landscape in male germ cells. In this study, our data demonstrated the presence of R-loops in fully mature human and bonobo sperm heads and their partial correspondence to transcribed regions and chromatin structure, which is massively reorganized from mainly histone to mainly protamine-packed chromatin in mature sperm. The sperm R-loop landscape resembles characteristic patterns of somatic cells. Surprisingly, we detected R-loops in both residual histone and protamine-packed chromatin and localize them to still-active retroposons, ALUs and SINE-VNTR-ALUs (SVAs), the latter has recently arisen in hominoid primates. We detected both evolutionarily conserved and species-specific localizations. Comparing our DNA-RNA immunoprecipitation (DRIP) data with published DNA methylation and histone chromatin immunoprecipitation (ChIP) data, we hypothesize that R-loops epigenetically reduce methylation of SVAs. Strikingly, we observe a strong influence of R-loops on the transcriptomes of zygotes from early developmental stages before zygotic genome activation. Overall, these findings suggest that chromatin accessibility influenced by R-loops may represent a system of inherited gene regulation.

In myeloid malignancies including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), patient selection and therapeutic strategies are increasingly based on tumor-specific genetic mutations. Among these, mutations in DDX41, which encodes a DEAD-box type RNA helicase, are present in approximately 2-5% of AML and MDS patients; this disease subtype exhibits a distinctive disease phenotype characterized by late age of onset, tendency toward cytopenia in the peripheral blood and bone marrow, a relatively favorable prognosis, and a high frequency of normal karyotypes. Typically, individuals with a loss-of-function germline DDX41 variant in one allele later acquire the p.R525H mutation in the other allele before overt disease manifestation, suggesting that the progressive decrease in DDX41 expression and/or function is involved in myeloid leukemogenesis. RNA helicases play roles in many processes involving RNA metabolism by altering RNA structure and RNA-protein interactions through ATP-dependent helicase activity. A single RNA helicase can play multiple cellular roles, making it difficult to elucidate the mechanisms by which mutations in DDX41 are involved in leukemogenesis. Nevertheless, multiple DDX41 functions have been associated with disease development. The enzyme has been implicated in the regulation of RNA splicing, nucleic acid sensing in the cytoplasm, R-loop resolution, and snoRNA processing. Most of the mutated RNA splicing-related factors in MDS are involved in the recognition and determination of 3' splice sites (SS), although their individual roles are distinct. On the other hand, DDX41 is likely incorporated into the C complex of the spliceosome, which may define a distinctive disease phenotype. This review summarizes the current understanding of how DDX41 is involved in this unique myeloid malignancy. [ABSTRACT FROM AUTHOR]

Fragile X-associated tremor/ataxia syndrome (FXTAS) and fragile X syndrome (FXS) are primary examples of fragile X-related disorders (FXDs) caused by abnormal expansion of CGG repeats above a certain threshold in the 5'-untranslated region of the fragile X mental retardation (FMR1) gene. Both diseases have distinct clinical manifestations and molecular pathogenesis. FXTAS is a late-adult-onset neurodegenerative disorder caused by a premutation (PM) allele (CGG expansion of 55-200 repeats), resulting in FMR1 gene hyperexpression. On the other hand, FXS is a neurodevelopmental disorder that results from a full mutation (FM) allele (CGG expansions of =200 repeats) leading to heterochromatization and transcriptional silencing of the FMR1 gene. The main challenge is to determine how CGG repeat expansion affects the fundamentally distinct nature of FMR1 expression in FM and PM ranges. Abnormal CGG repeat expansions form a variety of non-canonical DNA and RNA structures that can disrupt various cellular processes and cause distinct effects in PM and FM alleles. Here, we review these structures and how they are related to underlying mutations and disease pathology in FXS and FXTAS. Finally, as new CGG expansions within the genome have been identified, it will be interesting to determine their implications in disease pathology and treatment. [ABSTRACT FROM AUTHOR]

Fragile X-associated tremor/ataxia syndrome (FXTAS) and fragile X syndrome (FXS) are primary examples of fragile X-related disorders (FXDs) caused by abnormal expansion of CGG repeats above a certain threshold in the 5′-untranslated region of the fragile X mental retardation (FMR1) gene. Both diseases have distinct clinical manifestations and molecular pathogenesis. FXTAS is a late-adult-onset neurodegenerative disorder caused by a premutation (PM) allele (CGG expansion of 55–200 repeats), resulting in FMR1 gene hyperexpression. On the other hand, FXS is a neurodevelopmental disorder that results from a full mutation (FM) allele (CGG expansions of ≥200 repeats) leading to heterochromatization and transcriptional silencing of the FMR1 gene. The main challenge is to determine how CGG repeat expansion affects the fundamentally distinct nature of FMR1 expression in FM and PM ranges. Abnormal CGG repeat expansions form a variety of non-canonical DNA and RNA structures that can disrupt various cellular processes and cause distinct effects in PM and FM alleles. Here, we review these structures and how they are related to underlying mutations and disease pathology in FXS and FXTAS. Finally, as new CGG expansions within the genome have been identified, it will be interesting to determine their implications in disease pathology and treatment.

Telomere shortening rates inversely correlate with life expectancy and hence it is critical to understand how telomere shortening is regulated. Recently, the telomeric non-coding RNA, TERRA has been implicated in the regulation of replicative senescence. To better understand how TERRA is regulated we employed a proteomics approach to look for potential RNA regulators that associate with telomeric sequences. Based on the results, we have identified proteins that may regulate TERRA in both a positive and negative manner, depending on the state of the telomere. In this mini-review, we discuss and speculate about these data to expand our understanding of TERRA and telomere interactors with respect to telomere shortening dynamics. [ABSTRACT FROM AUTHOR]

Telomere shortening rates inversely correlate with life expectancy and hence it is critical to understand how telomere shortening is regulated. Recently, the telomeric non-coding RNA, TERRA has been implicated in the regulation of replicative senescence. To better understand how TERRA is regulated we employed a proteomics approach to look for potential RNA regulators that associate with telomeric sequences. Based on the results, we have identified proteins that may regulate TERRA in both a positive and negative manner, depending on the state of the telomere. In this mini-review, we discuss and speculate about these data to expand our understanding of TERRA and telomere interactors with respect to telomere shortening dynamics.

Over the last decades, numerous examples have involved nuclear non-coding RNAs (ncRNAs) in the regulation of gene expression. ncRNAs can interact with the genome by forming non-canonical nucleic acid structures such as R-loops or DNA:RNA triplexes. They bind chromatin and DNA modifiers and transcription factors and favor or prevent their targeting to specific DNA sequences and regulate gene expression of diverse genes. We review the function of these non-canonical nucleic acid structures in regulating gene expression of multicellular organisms during development and in response to different stress conditions and DNA damage using examples described in several organisms, from plants to humans. We also overview recent techniques developed to study where R-loops or DNA:RNA triplexes are formed in the genome and their interaction with proteins. [ABSTRACT FROM AUTHOR]

Over the last decades, numerous examples have involved nuclear non-coding RNAs (ncRNAs) in the regulation of gene expression. ncRNAs can interact with the genome by forming non-canonical nucleic acid structures such as R-loops or DNA:RNA triplexes. They bind chromatin and DNA modifiers and transcription factors and favor or prevent their targeting to specific DNA sequences and regulate gene expression of diverse genes. We review the function of these non-canonical nucleic acid structures in regulating gene expression of multicellular organisms during development and in response to different stress conditions and DNA damage using examples described in several organisms, from plants to humans. We also overview recent techniques developed to study where R-loops or DNA:RNA triplexes are formed in the genome and their interaction with proteins.

Chromatin-enriched RNAs (cheRNAs) constitute a special class of long noncoding RNAs (lncRNAs) that are enriched around chromatin and function to activate neighboring or distal gene transcription. Recent studies have shown that cheRNAs affect chromatin structure and gene expression by recruiting chromatin modifiers or acting as bridges between distal enhancers and promoters. The abnormal transcription of cheRNAs plays an important role in the occurrence of many diseases, particularly tumors. The critical effect of cancer stem cells (CSCs) on the formation and development of tumors is well known, but the function of cheRNAs in tumorigenesis, especially in CSC proliferation and stemness maintenance, is not yet fully understood. This review focuses on the mechanisms of cheRNAs in epigenetic regulation and chromatin conformation and discusses the way cheRNAs function in CSCs to deepen the understanding of tumorigenesis and provide novel insight to advance tumor-targeting therapy.

The correct repair of DNA double-strand breaks is essential for maintaining the stability of the genome, thus ensuring the survival and fitness of any living organism. Indeed, the repair of these lesions is a complicated affair, in which several pathways compete for the DNA ends in a complex balance. Thus, the fine-tuning of the DNA double-strand break repair pathway choice relies on the different regulatory layers that respond to environmental cues. Among those different tiers of regulation, RNA modifications have just emerged as a promising field.

The nucleotide alarmone (p)ppGpp, signaling the stringent response, is known for more than 5 decades. The cellular turnover of the alarmone is regulated by RelA/SpoT homolog (RSH) superfamily of enzymes. There are long RSHs (RelA, SpoT, and Rel) and short RSHs [small alarmone synthetases (SAS) and small alarmone hydrolases (SAH)]. Long RSHs are multidomain proteins with (p)ppGpp synthesis, hydrolysis, and regulatory functions. Short RSHs are single-domain proteins with a single (p)ppGpp synthesis/hydrolysis function with few exceptions having two domains. Mycobacterial RelZ is a dual-domain SAS with RNase HII and the (p)ppGpp synthetase activity. SAS is known to impact multiple cellular functions independently and in accordance with the long RSH. Few SAS in bacteria including RelZ synthesize pGpp, the third small alarmone, along with the conventional (p)ppGpp. SAS can act as an RNA-binding protein for the negative allosteric inhibition of (p)ppGpp synthesis. Here, we initially recap the important features and molecular functions of different SAS that are previously characterized to understand the obligation for the “alarmone pool” produced by the long and short RSHs. Then, we focus on the RelZ, especially the combined functions of RNase HII and (p)ppGpp synthesis from a single polypeptide to connect with the recent findings of SAS as an RNA-binding protein. Finally, we conclude with the possibilities of using single-stranded RNA (ssRNA) as an additional therapeutic strategy to combat the persistent infections by inhibiting the redundant (p)ppGpp synthetases.

The nucleotide alarmone (p)ppGpp, signaling the stringent response, is known for more than 5 decades. The cellular turnover of the alarmone is regulated by RelA/SpoT homolog (RSH) superfamily of enzymes. There are long RSHs (RelA, SpoT, and Rel) and short RSHs [small alarmone synthetases (SAS) and small alarmone hydrolases (SAH)]. Long RSHs are multidomain proteins with (p)ppGpp synthesis, hydrolysis, and regulatory functions. Short RSHs are single-domain proteins with a single (p)ppGpp synthesis/hydrolysis function with few exceptions having two domains. Mycobacterial RelZ is a dual-domain SAS with RNase HII and the (p)ppGpp synthetase activity. SAS is known to impact multiple cellular functions independently and in accordance with the long RSH. Few SAS in bacteria including RelZ synthesize pGpp, the third small alarmone, along with the conventional (p)ppGpp. SAS can act as an RNA-binding protein for the negative allosteric inhibition of (p)ppGpp synthesis. Here, we initially recap the important features and molecular functions of different SAS that are previously characterized to understand the obligation for the "alarmone pool" produced by the long and short RSHs. Then, we focus on the RelZ, especially the combined functions of RNase HII and (p)ppGpp synthesis from a single polypeptide to connect with the recent findings of SAS as an RNA-binding protein. Finally, we conclude with the possibilities of using single-stranded RNA (ssRNA) as an additional therapeutic strategy to combat the persistent infections by inhibiting the redundant (p)ppGpp synthetases. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lncRNAs) possess a diverse array of regulatory functions including activation and silencing of gene transcription, regulation of splicing, and coordinating epigenetic modifications. GATA3-AS1 is a divergent lncRNA gene neighboring GATA3. GATA3 is considered the master regulator of TH2 lineage commitment enabling TH2 effector cells to efficiently transcribe genes encoding cytokines IL-4, IL-5, and IL-13. Here, we show that the GATA3-AS1 lncRNA is selectively expressed under TH2 polarizing conditions and is necessary for efficient transcription of GATA3, IL5, and IL13 genes, while being sufficient for GATA3 transcription. GATA3-AS1 is required for formation of permissive chromatin marks, H3K27 acetylation and H3K4 di/tri-methylation, at the GATA3-AS1-GATA3 locus. Further, GATA3-AS1 binds components of the MLL methyltransferase and forms a DNA-RNA hybrid (R-loop) thus tethering the MLL methyltransferase to the gene locus. Our results indicate a novel regulatory function for a divergent lncRNA and provide new insight into the function of lncRNAs in T helper cell differentiation.

Long non-coding RNAs (lncRNAs) possess a diverse array of regulatory functions including activation and silencing of gene transcription, regulation of splicing, and coordinating epigenetic modifications. GATA3-AS1 is a divergent lncRNA gene neighboring GATA3. GATA3 is considered the master regulator of TH2 lineage commitment enabling TH2 effector cells to efficiently transcribe genes encoding cytokines IL-4, IL-5, and IL-13. Here, we show that the GATA3-AS1 lncRNA is selectively expressed under TH2 polarizing conditions and is necessary for efficient transcription of GATA3, IL5 , and IL13 genes, while being sufficient for GATA3 transcription. GATA3-AS1 is required for formation of permissive chromatin marks, H3K27 acetylation and H3K4 di/tri-methylation, at the GATA3-AS1-GATA3 locus. Further, GATA3-AS1 binds components of the MLL methyltransferase and forms a DNA-RNA hybrid (R-loop) thus tethering the MLL methyltransferase to the gene locus. Our results indicate a novel regulatory function for a divergent lncRNA and provide new insight into the function of lncRNAs in T helper cell differentiation. [ABSTRACT FROM AUTHOR]