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Ferroptosis, a form of regulated cell death, has emerged as a crucial process in diverse pathophysiological states, encompassing cancer, neurodegenerative ailments, and ischemia-reperfusion injury. The glutathione (GSH)dependent lipid peroxidation pathway, chiefly governed by glutathione peroxidase 4 (GPX4), assumes an essential part in driving ferroptosis. GPX4, as the principal orchestrator of ferroptosis, has garnered significant attention across cancer, cardiovascular, and neuroscience domains over the past decade. Noteworthy investigations have elucidated the indispensable functions of ferroptosis in numerous diseases, including tumorigenesis, wherein robust ferroptosis within cells can impede tumor advancement. Recent research has underscored the complex regulatory role of non-coding RNAs (ncRNAs) in regulating the GSH-GPX4 network, thus influencing cellular susceptibility to ferroptosis. This exhaustive review endeavors to probe into the multifaceted processes by which ncRNAs control the GSH-GPX4 network in ferroptosis. Specifically, we delve into the functions of miRNAs, lncRNAs, and circRNAs in regulating GPX4 expression and impacting cellular susceptibility to ferroptosis. Moreover, we discuss the clinical implications of dysregulated interactions between ncRNAs and GPX4 in several conditions, underscoring their capacity as viable targets for therapeutic intervention. Additionally, the review explores emerging strategies aimed at targeting ncRNAs to modulate the GSHGPX4 pathway and manipulate ferroptosis for therapeutic advantage. A comprehensive understanding of these intricate regulatory networks furnishes insights into innovative therapeutic avenues for diseases associated with perturbed ferroptosis, thereby laying the groundwork for therapeutic interventions targeting ncRNAs in ferroptosis-related pathological conditions.

Ferroptosis, a form of regulated cell death, has emerged as a crucial process in diverse pathophysiological states, encompassing cancer, neurodegenerative ailments, and ischemia-reperfusion injury. The glutathione (GSH)dependent lipid peroxidation pathway, chiefly governed by glutathione peroxidase 4 (GPX4), assumes an essential part in driving ferroptosis. GPX4, as the principal orchestrator of ferroptosis, has garnered significant attention across cancer, cardiovascular, and neuroscience domains over the past decade. Noteworthy investigations have elucidated the indispensable functions of ferroptosis in numerous diseases, including tumorigenesis, wherein robust ferroptosis within cells can impede tumor advancement. Recent research has underscored the complex regulatory role of non-coding RNAs (ncRNAs) in regulating the GSH-GPX4 network, thus influencing cellular susceptibility to ferroptosis. This exhaustive review endeavors to probe into the multifaceted processes by which ncRNAs control the GSH-GPX4 network in ferroptosis. Specifically, we delve into the functions of miRNAs, lncRNAs, and circRNAs in regulating GPX4 expression and impacting cellular susceptibility to ferroptosis. Moreover, we discuss the clinical implications of dysregulated interactions between ncRNAs and GPX4 in several conditions, underscoring their capacity as viable targets for therapeutic intervention. Additionally, the review explores emerging strategies aimed at targeting ncRNAs to modulate the GSHGPX4 pathway and manipulate ferroptosis for therapeutic advantage. A comprehensive understanding of these intricate regulatory networks furnishes insights into innovative therapeutic avenues for diseases associated with perturbed ferroptosis, thereby laying the groundwork for therapeutic interventions targeting ncRNAs in ferroptosis-related pathological conditions.

Ferroptosis, a form of regulated cell death, has emerged as a crucial process in diverse pathophysiological states, encompassing cancer, neurodegenerative ailments, and ischemia-reperfusion injury. The glutathione (GSH)dependent lipid peroxidation pathway, chiefly governed by glutathione peroxidase 4 (GPX4), assumes an essential part in driving ferroptosis. GPX4, as the principal orchestrator of ferroptosis, has garnered significant attention across cancer, cardiovascular, and neuroscience domains over the past decade. Noteworthy investigations have elucidated the indispensable functions of ferroptosis in numerous diseases, including tumorigenesis, wherein robust ferroptosis within cells can impede tumor advancement. Recent research has underscored the complex regulatory role of non-coding RNAs (ncRNAs) in regulating the GSH-GPX4 network, thus influencing cellular susceptibility to ferroptosis. This exhaustive review endeavors to probe into the multifaceted processes by which ncRNAs control the GSH-GPX4 network in ferroptosis. Specifically, we delve into the functions of miRNAs, lncRNAs, and circRNAs in regulating GPX4 expression and impacting cellular susceptibility to ferroptosis. Moreover, we discuss the clinical implications of dysregulated interactions between ncRNAs and GPX4 in several conditions, underscoring their capacity as viable targets for therapeutic intervention. Additionally, the review explores emerging strategies aimed at targeting ncRNAs to modulate the GSHGPX4 pathway and manipulate ferroptosis for therapeutic advantage. A comprehensive understanding of these intricate regulatory networks furnishes insights into innovative therapeutic avenues for diseases associated with perturbed ferroptosis, thereby laying the groundwork for therapeutic interventions targeting ncRNAs in ferroptosis-related pathological conditions.

Ferroptosis, a form of regulated cell death, has emerged as a crucial process in diverse pathophysiological states, encompassing cancer, neurodegenerative ailments, and ischemia-reperfusion injury. The glutathione (GSH)dependent lipid peroxidation pathway, chiefly governed by glutathione peroxidase 4 (GPX4), assumes an essential part in driving ferroptosis. GPX4, as the principal orchestrator of ferroptosis, has garnered significant attention across cancer, cardiovascular, and neuroscience domains over the past decade. Noteworthy investigations have elucidated the indispensable functions of ferroptosis in numerous diseases, including tumorigenesis, wherein robust ferroptosis within cells can impede tumor advancement. Recent research has underscored the complex regulatory role of non-coding RNAs (ncRNAs) in regulating the GSH-GPX4 network, thus influencing cellular susceptibility to ferroptosis. This exhaustive review endeavors to probe into the multifaceted processes by which ncRNAs control the GSH-GPX4 network in ferroptosis. Specifically, we delve into the functions of miRNAs, lncRNAs, and circRNAs in regulating GPX4 expression and impacting cellular susceptibility to ferroptosis. Moreover, we discuss the clinical implications of dysregulated interactions between ncRNAs and GPX4 in several conditions, underscoring their capacity as viable targets for therapeutic intervention. Additionally, the review explores emerging strategies aimed at targeting ncRNAs to modulate the GSHGPX4 pathway and manipulate ferroptosis for therapeutic advantage. A comprehensive understanding of these intricate regulatory networks furnishes insights into innovative therapeutic avenues for diseases associated with perturbed ferroptosis, thereby laying the groundwork for therapeutic interventions targeting ncRNAs in ferroptosis-related pathological conditions.

Ferroptosis, a form of regulated cell death, has emerged as a crucial process in diverse pathophysiological states, encompassing cancer, neurodegenerative ailments, and ischemia-reperfusion injury. The glutathione (GSH)dependent lipid peroxidation pathway, chiefly governed by glutathione peroxidase 4 (GPX4), assumes an essential part in driving ferroptosis. GPX4, as the principal orchestrator of ferroptosis, has garnered significant attention across cancer, cardiovascular, and neuroscience domains over the past decade. Noteworthy investigations have elucidated the indispensable functions of ferroptosis in numerous diseases, including tumorigenesis, wherein robust ferroptosis within cells can impede tumor advancement. Recent research has underscored the complex regulatory role of non-coding RNAs (ncRNAs) in regulating the GSH-GPX4 network, thus influencing cellular susceptibility to ferroptosis. This exhaustive review endeavors to probe into the multifaceted processes by which ncRNAs control the GSH-GPX4 network in ferroptosis. Specifically, we delve into the functions of miRNAs, lncRNAs, and circRNAs in regulating GPX4 expression and impacting cellular susceptibility to ferroptosis. Moreover, we discuss the clinical implications of dysregulated interactions between ncRNAs and GPX4 in several conditions, underscoring their capacity as viable targets for therapeutic intervention. Additionally, the review explores emerging strategies aimed at targeting ncRNAs to modulate the GSHGPX4 pathway and manipulate ferroptosis for therapeutic advantage. A comprehensive understanding of these intricate regulatory networks furnishes insights into innovative therapeutic avenues for diseases associated with perturbed ferroptosis, thereby laying the groundwork for therapeutic interventions targeting ncRNAs in ferroptosis-related pathological conditions.

Atherosclerosis and non-alcoholic fatty liver disease (NAFLD) are pathologies related to ectopic fat accumulation, both of which are continuously increasing in prevalence. These threats are prompting researchers to develop effective therapies for their clinical management. One of the common pathophysiological alterations that underlies both diseases is oxidative stress (OxS), which appears as a result of lipid deposition in affected tissues. However, the molecular mechanisms that lead to OxS generation are different in each disease. Non-coding RNAs (ncRNAs) are RNA transcripts that do not encode proteins and function by regulating gene expression. In recent years, the involvement of ncRNAs in OxS modulation has become more recognized. This review summarizes the most recent advances regarding ncRNA-mediated regulation of OxS in atherosclerosis and NAFLD. In both diseases, ncRNAs can exert pro-oxidant or antioxidant functions by regulating gene targets and even other ncRNAs, positioning them as potential therapeutic targets. Interestingly, both diseases have common altered ncRNAs, suggesting that the same molecule can be targeted simultaneously when both diseases coexist. Finally, since some ncRNAs have already been used as therapeutic agents, their roles as potential drugs for the clinical management of atherosclerosis and NAFLD are analyzed., Ministerio de Ciencia e Innovación (España), Depto. de Bioquímica y Biología Molecular, Fac. de Farmacia, TRUE, pub, Descuento UCM

RNA, DNA, and protein molecules are highly organized within three-dimensional (3D) structures in the nucleus. Although RNA has been proposed to play a role in nuclear organization, exploring this has been challenging because existing methods cannot measure higher-order RNA and DNA contacts within 3D structures. To address this, we developed RNA & DNA SPRITE (RD-SPRITE) to comprehensively map the spatial organization of RNA and DNA. These maps reveal higher-order RNA-chromatin structures associated with three major classes of nuclear function: RNA processing, heterochromatin assembly, and gene regulation. These data demonstrate that hundreds of ncRNAs form high-concentration territories throughout the nucleus, that specific RNAs are required to recruit various regulators into these territories, and that these RNAs can shape long-range DNA contacts, heterochromatin assembly, and gene expression. These results demonstrate a mechanism where RNAs form high-concentration territories, bind to diffusible regulators, and guide them into compartments to regulate essential nuclear functions.

Long non-coding RNAs (lncRNAs) can perform a variety of key cellular functions by interacting with proteins and other RNAs. Recent studies have shown that the functions of lncRNAS are largely mediated by their structures. However, our structural knowledge for most lncRNAS is limited to sequence-based computational predictions. Non-coding RNA activated by DNA damage (NORAD) is an atypical lncRNA due to its abundant expression and high sequence conservation. NORAD regulates genomic stability by interacting with proteins and microRNAs. Previous sequence-based characterization has identified a modular organization of NORAD composed of several NORAD repeat units (NRUs). These units comprise the protein-binding elements and are separated by regular spacers. Here, we experimentally determine for the first time the secondary structure of NORAD using the nextPARS approach. Our results suggest that the spacer regions provide structural stability to NRUs. Furthermore, we uncover two previously unreported NRUs, and determine the core structural motifs conserved across NRUs. Overall, these findings will help to elucidate the function and evolution of NORAD., UC was funded in part through H2020 Marie Skłodowska-Curie Actions (H2020-MSCA-IF-2017-793699) and MICINN (IJC2019-039402-I). TG group acknowledges support from the Spanish Ministry of Science and Innovation for grant PGC2018-099921-B-I00, cofounded by European Regional Development Fund (ERDF); from the Catalan Research Agency (AGAUR) SGR423; from the European Union’s Horizon 2020 research and innovation programme (ERC-2016-724173); from the Gordon and Betty Moore Foundation (Grant GBMF9742) and from the Instituto de Salud Carlos III (INB Grant PT17/0009/0023 - ISCIII-SGEFI/ERDF)., Peer Reviewed, Postprint (published version)

The mammalian genome comprehends a small minority of genes that encode for proteins (barely 2% of the total genome in humans) and an immense majority of genes that are transcribed into RNA but not encoded for proteins (ncRNAs). These non-coding genes are intimately related to the expression regulation of protein-coding genes. The ncRNAs subtypes differ in their size, so there are long non-coding genes (lncRNAs) and other smaller ones, like microRNAs (miRNAs) and piwi-interacting RNAs (piRNAs). Due to their important role in the maintenance of cellular functioning, any deregulation of the expression profiles of these ncRNAs can dissemble in the development of different types of diseases. Among them, we can highlight some of high incidence in the population, such as cancer, neurodegenerative, or cardiovascular disorders. In addition, thanks to the enormous advances in the field of medical genomics, these same ncRNAs are starting to be used as possible drugs, approved by the FDA, as an effective treatment for diseases

B chromosomes are enigmatic heritable elements found in the genomes of numerous plant and animal species. Contrary to their broad distribution, most B chromosomes are non-essential. For this reason, they are regarded as genome parasites. In order to be stably transmitted through generations, many B chromosomes exhibit the ability to "drive", i.e., they transmit themselves at super-Mendelian frequencies to progeny through directed interactions with the cell division apparatus. To date, very little is understood mechanistically about how B chromosomes drive, although a likely scenario is that expression of B chromosome sequences plays a role. Here, we highlight a handful of previously identified B chromosome sequences, many of which are repetitive and non-coding in nature, that have been shown to be expressed at the transcriptional level. We speculate on how each type of expressed sequence could participate in B chromosome drive based on known functions of RNA in general chromatin- and chromosome-related processes. We also raise some challenges to functionally testing these possible roles, a goal that will be required to more fully understand whether and how B chromosomes interact with components of the cell for drive and transmission.

[EN] Small interfering RNAs (siRNA) are key regulators of gene expression that play essential roles in diverse biological processes. Trans-acting siRNAs (tasiRNAs) are a class of plant-endogenous siRNAs that lead the cleavage of nonidentical transcripts. TasiRNAs are usually involved in fine-tuning development. However, increasing evidence supports that tasiRNAs may be involved in stress response. Melon is a crop of great economic importance extensively cultivated in semiarid regions frequently exposed to changing environmental conditions that limit its productivity. However, knowledge of the precise role of siRNAs in general, and of tasiRNAs in particular, in regulating the response to adverse environmental conditions is limited. Here, we provide the first comprehensive analysis of computationally inferred melon-tasiRNAs responsive to two biotic (viroid-infection) and abiotic (cold treatment) stress conditions. We identify two TAS3-loci encoding to length (TAS3-L) and short (TAS3-S) transcripts. The TAS candidates predicted from small RNA-sequencing data were characterized according to their chromosome localization and expression pattern in response to stress. The functional activity of cmTAS genes was validated by transcript quantification and degradome assays of the tasiRNA precursors and their predicted targets. Finally, the functionality of a representative cmTAS3-derived tasiRNA (TAS3-S) was confirmed by transient assays showing the cleavage of ARF target transcripts.

B chromosomes are enigmatic heritable elements found in the genomes of numerous plant and animal species. Contrary to their broad distribution, most B chromosomes are non-essential. For this reason, they are regarded as genome parasites. In order to be stably transmitted through generations, many B chromosomes exhibit the ability to "drive", i.e., they transmit themselves at super-Mendelian frequencies to progeny through directed interactions with the cell division apparatus. To date, very little is understood mechanistically about how B chromosomes drive, although a likely scenario is that expression of B chromosome sequences plays a role. Here, we highlight a handful of previously identified B chromosome sequences, many of which are repetitive and non-coding in nature, that have been shown to be expressed at the transcriptional level. We speculate on how each type of expressed sequence could participate in B chromosome drive based on known functions of RNA in general chromatin- and chromosome-related processes. We also raise some challenges to functionally testing these possible roles, a goal that will be required to more fully understand whether and how B chromosomes interact with components of the cell for drive and transmission.

[EN] Multiple sclerosis (MS) is a common inflammatory and neurodegenerative disease that causes neurological disability. Transcriptome regulation has been seen affected in MS patients’ blood cells. Recently, non-coding RNAs (ncRNAs) have emerged as prominent transcriptome regulators, and in turn, they could play an important role in MS pathogenesis. From this point of view, the multiple sclerosis group in IIS BioDonostia has already analyzed the expression of protein-coding RNAs and some ncRNAs, especially miRNAs, in search of putative MS-related genes. These studies highlighted some possible candidates, amongst others, the snoRNA SNORA40. Thus, the aim of this work is to analyze snoRNAs located in MS-associated regions of the genome and further investigate the possible MS relationship of SNORA40. For that purpose, after detecting the candidate snoRNAs and optimizing the conditions for amplifying them, we did the sequencing of 10 snoRNAs in 14 siblings from 7 Gipuzkoan families and the sequencing of SNORA40 in 47 samples previously included in a RNA expression study. That way, we could identify both already described and novel genetic variants in some of the snoRNAs, but could not relate those variants to the disease as to our samples, ruling out a possible Mendelian inheritance way of those genetic variants.

[EN] Multiple sclerosis (MS) is a common inflammatory and neurodegenerative disease that causes neurological disability. Transcriptome regulation has been seen affected in MS patients’ blood cells. Recently, non-coding RNAs (ncRNAs) have emerged as prominent transcriptome regulators, and in turn, they could play an important role in MS pathogenesis. From this point of view, the multiple sclerosis group in IIS BioDonostia has already analyzed the expression of protein-coding RNAs and some ncRNAs, especially miRNAs, in search of putative MS-related genes. These studies highlighted some possible candidates, amongst others, the snoRNA SNORA40. Thus, the aim of this work is to analyze snoRNAs located in MS-associated regions of the genome and further investigate the possible MS relationship of SNORA40. For that purpose, after detecting the candidate snoRNAs and optimizing the conditions for amplifying them, we did the sequencing of 10 snoRNAs in 14 siblings from 7 Gipuzkoan families and the sequencing of SNORA40 in 47 samples previously included in a RNA expression study. That way, we could identify both already described and novel genetic variants in some of the snoRNAs, but could not relate those variants to the disease as to our samples, ruling out a possible Mendelian inheritance way of those genetic variants.

[EN] Multiple sclerosis (MS) is a common inflammatory and neurodegenerative disease that causes neurological disability. Transcriptome regulation has been seen affected in MS patients’ blood cells. Recently, non-coding RNAs (ncRNAs) have emerged as prominent transcriptome regulators, and in turn, they could play an important role in MS pathogenesis. From this point of view, the multiple sclerosis group in IIS BioDonostia has already analyzed the expression of protein-coding RNAs and some ncRNAs, especially miRNAs, in search of putative MS-related genes. These studies highlighted some possible candidates, amongst others, the snoRNA SNORA40. Thus, the aim of this work is to analyze snoRNAs located in MS-associated regions of the genome and further investigate the possible MS relationship of SNORA40. For that purpose, after detecting the candidate snoRNAs and optimizing the conditions for amplifying them, we did the sequencing of 10 snoRNAs in 14 siblings from 7 Gipuzkoan families and the sequencing of SNORA40 in 47 samples previously included in a RNA expression study. That way, we could identify both already described and novel genetic variants in some of the snoRNAs, but could not relate those variants to the disease as to our samples, ruling out a possible Mendelian inheritance way of those genetic variants.

[EN] Lung cancer is a highly lethal disease. Targeted therapies have been developed in last years, however survival rates are not improving due to the delay in the diagnosis, making biomarkers one of the most interesting fields of study in cancer. Liquid biopsy has raised as an alternative to tissue biopsy due to improvements in analytical techniques for circulating tumor cells (CTCs), cell free DNA and exosomes. Among all, exosomes have raised as one of the most promising tools to understand the tumor due to their stability in the blood and their similarity to the cells of origin. In the last years, different alterations have been described inside the exosomes derived from non-small cell lung cancer (NSCLC) cells mirroring the processes inside these tumoral cells, such as EGFR mutation, translocations or microRNA (miRNA) deregulation. All these studies have opened the window to a new world of possibilities in the study of tumor biomarkers.

[EN] Lung cancer is a highly lethal disease. Targeted therapies have been developed in last years, however survival rates are not improving due to the delay in the diagnosis, making biomarkers one of the most interesting fields of study in cancer. Liquid biopsy has raised as an alternative to tissue biopsy due to improvements in analytical techniques for circulating tumor cells (CTCs), cell free DNA and exosomes. Among all, exosomes have raised as one of the most promising tools to understand the tumor due to their stability in the blood and their similarity to the cells of origin. In the last years, different alterations have been described inside the exosomes derived from non-small cell lung cancer (NSCLC) cells mirroring the processes inside these tumoral cells, such as EGFR mutation, translocations or microRNA (miRNA) deregulation. All these studies have opened the window to a new world of possibilities in the study of tumor biomarkers.

Human exposure to nanomaterials and nanoparticles is increasing rapidly, but their effects on human health are still largely unknown. Epigenetic modifications are attracting ever more interest as possible underlying molecular mechanisms of gene–environment interactions, highlighting them as potential molecular targets following exposure to nanomaterials and nanoparticles. Interestingly, recent research has identified changes in DNA methylation, histone post-translational modifications, and noncoding RNAs in mammalian cells exposed to nanomaterials and nanoparticles. However, the challenge for the future will be to determine the molecular pathways driving these epigenetic alterations, the possible functional consequences, and the potential effects on health.

1, PAGANI, MASSIMILIANO, 4415, open, Long non-coding-RNAs are emerging as important regulators of cellular functions but little is known on their role in human immune system. Here we investigated long intergenic non-coding-RNAs (lincRNAs) in TH1 lymphocyte subset. We focused on the TH1-specific lincRNA linc-MAF-4, whose gene is located ≈140 kb upstream MAF: a TH2-associated transcription factor. Linc-MAF-4 expression was found to anti-correlate with MAF. Also, down-regulation of linc-MAF-4 increases the expression of TH2-specific genes and skews T cell differentiation toward TH2 fate. Linc-MAF-4 is enriched in the chromatin fraction and thanks to a long-distance chromosome interaction between linc-MAF-4 and MAF genomic regions, linc-MAF-4 recruits LSD1 and EZH2 at MAF promoter to downregulate its transcription. These data demonstrate a key role of lincRNAs in human T lymphocyte differentiation., No, embargoed_20171127, Panzeri, I, Panzeri, I

Non-coding RNAs (ncRNAs) have diverse essential biological functions in all organisms, and in eukaryotes, two such classes of ncRNAs are the small nucleolar (sno) and small nuclear (sn) RNAs. In this study, we have identified and characterized a collection of sno and snRNAs in Giardia lamblia, by exploiting our discovery of a conserved 12 nt RNA processing sequence motif found in the 30 end regions of a large number of G. lamblia ncRNA genes. RNA end mapping and other experiments indicate the motif serves to mediate ncRNA 30 end formation from mono- and di-cistronic RNA precursor transcripts. Remarkably, we find the motif is also utilized in the processing pathway of all four previously identified trans-spliced G. lamblia introns, revealing a common RNA processing pathway for ncRNAs and trans-spliced introns in this organism. Motif sequence conservation then allowed for the bioinformatic and experimental identification of additional G. lamblia ncRNAs, including new U1 and U6 spliceosomal snRNA candidates. The U6 snRNA candidate was then used as a tool to identity novel U2 and U4 snRNAs, based on predicted phylogenetically conserved snRNA–snRNA base-pairing interactions, from a set of previously identified G. lamblia ncRNAs without assigned function. The Giardia snRNAs retain the core features of spliceosomal snRNAs but are sufficiently evolutionarily divergent to explain the difficulties in their identification. Most intriguingly, all of these snRNAs show structural features diagnostic of U2-dependent/major and U12-dependent/minor spliceosomal snRNAs.

Non-coding RNAs (ncRNAs) have diverse essential biological functions in all organisms, and in eukaryotes, two such classes of ncRNAs are the small nucleolar (sno) and small nuclear (sn) RNAs. In this study, we have identified and characterized a collection of sno and snRNAs in Giardia lamblia, by exploiting our discovery of a conserved 12 nt RNA processing sequence motif found in the 30 end regions of a large number of G. lamblia ncRNA genes. RNA end mapping and other experiments indicate the motif serves to mediate ncRNA 30 end formation from mono- and di-cistronic RNA precursor transcripts. Remarkably, we find the motif is also utilized in the processing pathway of all four previously identified trans-spliced G. lamblia introns, revealing a common RNA processing pathway for ncRNAs and trans-spliced introns in this organism. Motif sequence conservation then allowed for the bioinformatic and experimental identification of additional G. lamblia ncRNAs, including new U1 and U6 spliceosomal snRNA candidates. The U6 snRNA candidate was then used as a tool to identity novel U2 and U4 snRNAs, based on predicted phylogenetically conserved snRNA–snRNA base-pairing interactions, from a set of previously identified G. lamblia ncRNAs without assigned function. The Giardia snRNAs retain the core features of spliceosomal snRNAs but are sufficiently evolutionarily divergent to explain the difficulties in their identification. Most intriguingly, all of these snRNAs show structural features diagnostic of U2-dependent/major and U12-dependent/minor spliceosomal snRNAs.

Non-coding RNAs (ncRNAs) have diverse essential biological functions in all organisms, and in eukaryotes, two such classes of ncRNAs are the small nucleolar (sno) and small nuclear (sn) RNAs. In this study, we have identified and characterized a collection of sno and snRNAs in Giardia lamblia, by exploiting our discovery of a conserved 12 nt RNA processing sequence motif found in the 30 end regions of a large number of G. lamblia ncRNA genes. RNA end mapping and other experiments indicate the motif serves to mediate ncRNA 30 end formation from mono- and di-cistronic RNA precursor transcripts. Remarkably, we find the motif is also utilized in the processing pathway of all four previously identified trans-spliced G. lamblia introns, revealing a common RNA processing pathway for ncRNAs and trans-spliced introns in this organism. Motif sequence conservation then allowed for the bioinformatic and experimental identification of additional G. lamblia ncRNAs, including new U1 and U6 spliceosomal snRNA candidates. The U6 snRNA candidate was then used as a tool to identity novel U2 and U4 snRNAs, based on predicted phylogenetically conserved snRNA–snRNA base-pairing interactions, from a set of previously identified G. lamblia ncRNAs without assigned function. The Giardia snRNAs retain the core features of spliceosomal snRNAs but are sufficiently evolutionarily divergent to explain the difficulties in their identification. Most intriguingly, all of these snRNAs show structural features diagnostic of U2-dependent/major and U12-dependent/minor spliceosomal snRNAs.

Non-coding RNAs (ncRNAs) have diverse essential biological functions in all organisms, and in eukaryotes, two such classes of ncRNAs are the small nucleolar (sno) and small nuclear (sn) RNAs. In this study, we have identified and characterized a collection of sno and snRNAs in Giardia lamblia, by exploiting our discovery of a conserved 12 nt RNA processing sequence motif found in the 30 end regions of a large number of G. lamblia ncRNA genes. RNA end mapping and other experiments indicate the motif serves to mediate ncRNA 30 end formation from mono- and di-cistronic RNA precursor transcripts. Remarkably, we find the motif is also utilized in the processing pathway of all four previously identified trans-spliced G. lamblia introns, revealing a common RNA processing pathway for ncRNAs and trans-spliced introns in this organism. Motif sequence conservation then allowed for the bioinformatic and experimental identification of additional G. lamblia ncRNAs, including new U1 and U6 spliceosomal snRNA candidates. The U6 snRNA candidate was then used as a tool to identity novel U2 and U4 snRNAs, based on predicted phylogenetically conserved snRNA–snRNA base-pairing interactions, from a set of previously identified G. lamblia ncRNAs without assigned function. The Giardia snRNAs retain the core features of spliceosomal snRNAs but are sufficiently evolutionarily divergent to explain the difficulties in their identification. Most intriguingly, all of these snRNAs show structural features diagnostic of U2-dependent/major and U12-dependent/minor spliceosomal snRNAs.

The Gram-positive human pathogen Listeria monocytogenes uses a wide range of virulence factors for its pathogenesis. The majority of its virulence genes are encoded on a 9-kb pathogenicity island and are controlled by the transcriptional activator PrfA. Expression of these genes is maximal at 37°C and minimal at 30°C in a mechanism involving an RNA thermosensor. This thesis brings up different aspects of RNA-mediated regulation, including regulatory RNA structures within coding mRNA controlling expression to 5-untranslated RNA (5´-UTR) that controls downstream genes (cis-acting) as well as small non-coding RNAs (ncRNAs) that bind other target RNA (trans-acting). We investigated the importance of the coding region of the prfA-mRNA for its expression. Various lengths of prfA-mRNA were fused with reporter genes. Our finding suggested that the first 20 codons of prfA-mRNA were essential for efficient translation in Listeria monocytogenes. Translation of the shorter constructs was shown to be reduced. The expression level showed an inverse correlation with the RNA secondary structure stability in the beginning of the coding region. Riboswitches have previously been known to control expression of their downstream mRNA in a cis-acting manner. A trans-acting S-adenosylmethionine-binding riboswitch termed SreA was identified in Listeria monocytogenes. It was found to control the expression of the virulence regulator PrfA, by binding to the prfA-UTR and thereby affecting its translation. We examined the RNA locus encoding different virulence factors in Listeria monocytogenes. Several of them were preceded by 5´-UTRs of various lengths. We speculate that these 5´-UTRs could control expression of the downstream mRNA, provided they are of sufficient length. These findings prompted us to examine where and when Listeria monocytogenes switches on gene expression. Tiling array was used to compare RNAs isolated from wild-type and mutant bacteria grown at different growth conditions.

The Gram-positive human pathogen Listeria monocytogenes uses a wide range of virulence factors for its pathogenesis. The majority of its virulence genes are encoded on a 9-kb pathogenicity island and are controlled by the transcriptional activator PrfA. Expression of these genes is maximal at 37°C and minimal at 30°C in a mechanism involving an RNA thermosensor. This thesis brings up different aspects of RNA-mediated regulation, including regulatory RNA structures within coding mRNA controlling expression to 5-untranslated RNA (5´-UTR) that controls downstream genes (cis-acting) as well as small non-coding RNAs (ncRNAs) that bind other target RNA (trans-acting). We investigated the importance of the coding region of the prfA-mRNA for its expression. Various lengths of prfA-mRNA were fused with reporter genes. Our finding suggested that the first 20 codons of prfA-mRNA were essential for efficient translation in Listeria monocytogenes. Translation of the shorter constructs was shown to be reduced. The expression level showed an inverse correlation with the RNA secondary structure stability in the beginning of the coding region. Riboswitches have previously been known to control expression of their downstream mRNA in a cis-acting manner. A trans-acting S-adenosylmethionine-binding riboswitch termed SreA was identified in Listeria monocytogenes. It was found to control the expression of the virulence regulator PrfA, by binding to the prfA-UTR and thereby affecting its translation. We examined the RNA locus encoding different virulence factors in Listeria monocytogenes. Several of them were preceded by 5´-UTRs of various lengths. We speculate that these 5´-UTRs could control expression of the downstream mRNA, provided they are of sufficient length. These findings prompted us to examine where and when Listeria monocytogenes switches on gene expression. Tiling array was used to compare RNAs isolated from wild-type and mutant bacteria grown at different growth conditions.

The Gram-positive human pathogen Listeria monocytogenes uses a wide range of virulence factors for its pathogenesis. The majority of its virulence genes are encoded on a 9-kb pathogenicity island and are controlled by the transcriptional activator PrfA. Expression of these genes is maximal at 37°C and minimal at 30°C in a mechanism involving an RNA thermosensor. This thesis brings up different aspects of RNA-mediated regulation, including regulatory RNA structures within coding mRNA controlling expression to 5-untranslated RNA (5´-UTR) that controls downstream genes (cis-acting) as well as small non-coding RNAs (ncRNAs) that bind other target RNA (trans-acting). We investigated the importance of the coding region of the prfA-mRNA for its expression. Various lengths of prfA-mRNA were fused with reporter genes. Our finding suggested that the first 20 codons of prfA-mRNA were essential for efficient translation in Listeria monocytogenes. Translation of the shorter constructs was shown to be reduced. The expression level showed an inverse correlation with the RNA secondary structure stability in the beginning of the coding region. Riboswitches have previously been known to control expression of their downstream mRNA in a cis-acting manner. A trans-acting S-adenosylmethionine-binding riboswitch termed SreA was identified in Listeria monocytogenes. It was found to control the expression of the virulence regulator PrfA, by binding to the prfA-UTR and thereby affecting its translation. We examined the RNA locus encoding different virulence factors in Listeria monocytogenes. Several of them were preceded by 5´-UTRs of various lengths. We speculate that these 5´-UTRs could control expression of the downstream mRNA, provided they are of sufficient length. These findings prompted us to examine where and when Listeria monocytogenes switches on gene expression. Tiling array was used to compare RNAs isolated from wild-type and mutant bacteria grown at different growth conditions.

The Gram-positive human pathogen Listeria monocytogenes uses a wide range of virulence factors for its pathogenesis. The majority of its virulence genes are encoded on a 9-kb pathogenicity island and are controlled by the transcriptional activator PrfA. Expression of these genes is maximal at 37°C and minimal at 30°C in a mechanism involving an RNA thermosensor. This thesis brings up different aspects of RNA-mediated regulation, including regulatory RNA structures within coding mRNA controlling expression to 5-untranslated RNA (5´-UTR) that controls downstream genes (cis-acting) as well as small non-coding RNAs (ncRNAs) that bind other target RNA (trans-acting). We investigated the importance of the coding region of the prfA-mRNA for its expression. Various lengths of prfA-mRNA were fused with reporter genes. Our finding suggested that the first 20 codons of prfA-mRNA were essential for efficient translation in Listeria monocytogenes. Translation of the shorter constructs was shown to be reduced. The expression level showed an inverse correlation with the RNA secondary structure stability in the beginning of the coding region. Riboswitches have previously been known to control expression of their downstream mRNA in a cis-acting manner. A trans-acting S-adenosylmethionine-binding riboswitch termed SreA was identified in Listeria monocytogenes. It was found to control the expression of the virulence regulator PrfA, by binding to the prfA-UTR and thereby affecting its translation. We examined the RNA locus encoding different virulence factors in Listeria monocytogenes. Several of them were preceded by 5´-UTRs of various lengths. We speculate that these 5´-UTRs could control expression of the downstream mRNA, provided they are of sufficient length. These findings prompted us to examine where and when Listeria monocytogenes switches on gene expression. Tiling array was used to compare RNAs isolated from wild-type and mutant bacteria grown at different growth conditions.

Mehr als 75% des menschlichen Genoms wird von DNA in RNA transkribiert aber nur 2% kodieren für Proteine. Der größte Teil des Transkriptoms ist somit nicht protein-codierend und wird als non-coding RNA (ncRNA) bezeichnet. Eine Vielzahl von Studien konnte zeigen, dass viele dieser ncRNAs wichtige regulatorische Funktionen erfüllen. Viele dieser Funktionen werden mit unterschiedlichen Krankheiten in Verbindung gebracht. ncRNAs können in Tumorgenese, neurologischen Erkrankungen, Entwicklungsstörungen und anderen Erkrankungen eine entscheidende Rolle spielen und daher unterschiedlich exprimiert sein. Ein Hauptaugenmerk der diesbezüglichen RNA-Forschung lag bisher auf der Klasse der microRNAs (miRNA), welche die Genexpression auf unterschiedliche Weise beeinflussen können. Andere Klassen der ncRNAs wurden bis jetzt kaum oder nur wenig untersucht. Aus diesem Grund war das Ziel dieser Dissertation neue ncRNAs zu identifizieren und analysieren, die im Zusammenhang mit Multipler System Atrophy (MSA) und chronischer Niereninsuffizienz (CKD) stehen. Zur Identifikation differentiell exprimierter ncRNAs in MSA wurde RNA aus zwei verschiedenen Hirnregionen, Striatum und Substantia Nigra (SN), von genetisch veränderten Mäusen isoliert, die als Modelsystem für MSA gelten. Das Striatum und die SN, sind Gehirnareale, die im Verlauf der Krankheit starker Neurodegeneration unterliegen. Die RNA wurden mittels Microarray analysiert und die Ergebnisse bioinformatisch, basierend auf der R Bioconductor Plattform, im Speziellen mit Limma und MmPalateMiRNA, ausgewertet. Dadurch konnten 33 differentiell exprimierte miRNAs im Striatum und 59 miRNAs in SN identifiziert werden. Darüber hinaus wurden anhand von mRNA Sequenzierungen und Microarrays, die Expression von mRNAs untersucht. Die Zusammenhänge der Regulation neuer ncRNAs und den mRNAs wurde bioinformatisch analysiert und es konnten verschiedenste Prozesse identifiziert werden, welche eine entscheidende Rolle beim Verlauf von MSA spielen k, Recent transcriptome studies have elucidated that the human genome is pervasively transcribed and generates thousands of non-protein-coding RNA transcripts (ncRNAs), which are proposed to be involved in a variety of regulatory processes associated with human diseases. Thereby, several studies have shown that ncRNAs play important roles in tumorigenesis, neurological and developmental disorders, cardiovascular diseases, renal, kidney diseases or other disorders, respectively. To date, these studies have focused primarily on profiling microRNAs (miRNAs) expression patterns. However, not much is known about other classes of ncRNAs that are involved in human diseases. Therefore, work focused on the identification of regulatory ncRNAs, in addition to microRNAs, involved in two human diseases: Multiple System Atrophy (MSA) and Chronic Kidney Disease (CKD), respectively. To identify regulatory ncRNAs in an MSA mouse model, differential expression of ncRNAs was investigated by microarray analysis of substantia nigra (SN) and striatum, two brain regions that undergo neurodegeneration at a later stage of the disease in an MSA model. Thereby, in total, 33 differentially expressed microRNAs were identified in striatum and 59 miRNAs in SN. In addition, differential expression of mRNAs was bioinformatically analysed based on RNA-seq and mRNA microarray data from striatum and SN samples, respectively. Subsequently, miRNA-mRNA interactions were computationally predicted and were shown to be involved in specific biological processes linked to MSA. The negatively regulated miRNA-mRNA pairs were associated with immune system processes, in the very early non-symptomatic disease stages of MSA (SN and striatum). The early stages of MSA also showed association with homeostasis and oxidative stress in SN whereas protein handling, metabolism, transmembrane transport, cell death were linked to the striatum, respectively. To identify differentially expressed ncRNAs in CKD, expression analysis, MSc. Rimpi Khurana, Dissertation Medizinische Universität Innsbruck 2017

Mehr als 75% des menschlichen Genoms wird von DNA in RNA transkribiert aber nur 2% kodieren für Proteine. Der größte Teil des Transkriptoms ist somit nicht protein-codierend und wird als non-coding RNA (ncRNA) bezeichnet. Eine Vielzahl von Studien konnte zeigen, dass viele dieser ncRNAs wichtige regulatorische Funktionen erfüllen. Viele dieser Funktionen werden mit unterschiedlichen Krankheiten in Verbindung gebracht. ncRNAs können in Tumorgenese, neurologischen Erkrankungen, Entwicklungsstörungen und anderen Erkrankungen eine entscheidende Rolle spielen und daher unterschiedlich exprimiert sein. Ein Hauptaugenmerk der diesbezüglichen RNA-Forschung lag bisher auf der Klasse der microRNAs (miRNA), welche die Genexpression auf unterschiedliche Weise beeinflussen können. Andere Klassen der ncRNAs wurden bis jetzt kaum oder nur wenig untersucht. Aus diesem Grund war das Ziel dieser Dissertation neue ncRNAs zu identifizieren und analysieren, die im Zusammenhang mit Multipler System Atrophy (MSA) und chronischer Niereninsuffizienz (CKD) stehen. Zur Identifikation differentiell exprimierter ncRNAs in MSA wurde RNA aus zwei verschiedenen Hirnregionen, Striatum und Substantia Nigra (SN), von genetisch veränderten Mäusen isoliert, die als Modelsystem für MSA gelten. Das Striatum und die SN, sind Gehirnareale, die im Verlauf der Krankheit starker Neurodegeneration unterliegen. Die RNA wurden mittels Microarray analysiert und die Ergebnisse bioinformatisch, basierend auf der R Bioconductor Plattform, im Speziellen mit Limma und MmPalateMiRNA, ausgewertet. Dadurch konnten 33 differentiell exprimierte miRNAs im Striatum und 59 miRNAs in SN identifiziert werden. Darüber hinaus wurden anhand von mRNA Sequenzierungen und Microarrays, die Expression von mRNAs untersucht. Die Zusammenhänge der Regulation neuer ncRNAs und den mRNAs wurde bioinformatisch analysiert und es konnten verschiedenste Prozesse identifiziert werden, welche eine entscheidende Rolle beim Verlauf von MSA spielen k, Recent transcriptome studies have elucidated that the human genome is pervasively transcribed and generates thousands of non-protein-coding RNA transcripts (ncRNAs), which are proposed to be involved in a variety of regulatory processes associated with human diseases. Thereby, several studies have shown that ncRNAs play important roles in tumorigenesis, neurological and developmental disorders, cardiovascular diseases, renal, kidney diseases or other disorders, respectively. To date, these studies have focused primarily on profiling microRNAs (miRNAs) expression patterns. However, not much is known about other classes of ncRNAs that are involved in human diseases. Therefore, work focused on the identification of regulatory ncRNAs, in addition to microRNAs, involved in two human diseases: Multiple System Atrophy (MSA) and Chronic Kidney Disease (CKD), respectively. To identify regulatory ncRNAs in an MSA mouse model, differential expression of ncRNAs was investigated by microarray analysis of substantia nigra (SN) and striatum, two brain regions that undergo neurodegeneration at a later stage of the disease in an MSA model. Thereby, in total, 33 differentially expressed microRNAs were identified in striatum and 59 miRNAs in SN. In addition, differential expression of mRNAs was bioinformatically analysed based on RNA-seq and mRNA microarray data from striatum and SN samples, respectively. Subsequently, miRNA-mRNA interactions were computationally predicted and were shown to be involved in specific biological processes linked to MSA. The negatively regulated miRNA-mRNA pairs were associated with immune system processes, in the very early non-symptomatic disease stages of MSA (SN and striatum). The early stages of MSA also showed association with homeostasis and oxidative stress in SN whereas protein handling, metabolism, transmembrane transport, cell death were linked to the striatum, respectively. To identify differentially expressed ncRNAs in CKD, expression analysis, MSc. Rimpi Khurana, Dissertation Medizinische Universität Innsbruck 2017